Reprogramming of cells to a new fate

ABSTRACT

Methods and compositions for transdifferentiation of an animal cell from (i) a first pluripotent cell fate to a second nonpluripotent cell fate or (ii) from a non-pluripotent mesodermal, endodermal, or ectodermal cell fate to a different non-pluripotent mesodermal, endodermal, or ectodermal cell fate.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims benefit of priority to U.S. ProvisionalApplication No. 61/354,651, filed Jun. 14, 2010, U.S. ProvisionalApplication No. 61/376,148, filed Aug. 23, 2010, and U.S. ProvisionalApplication No. 61/437,294, filed Jan. 28, 2011, the contents of each ofwhich is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The mammalian heart lacks significant regenerative capacity. In vitrogeneration of cardiac cells in quantities sufficient for transplantationwould therefore revolutionize the treatment of heart disease. A recentreport of successful transdifferentiation of somatic cells to a cardiacfate in vitro (Ieda, M. et al., Cell 142, 375-86 (2010)) has raised thepossibility that this process might eventually be used for cell-basedcardiac therapy. However, speed and efficiency must first be improved,especially if the ultimate goal is to provide a faster and saferalternative to the re-differentiation of autologous induced pluripotentstem cells (iPSCs) (Schenke-Layland, K. et al., Stem Cells 26, 1537-46(2008)).

BRIEF SUMMARY OF THE INVENTION

The present invention provides for methods of converting an animal cellfrom a first non-pluripotent cell fate to a second non-pluripotent cellfate. In some embodiments, the method comprises:

-   -   increasing the quantity of at least one reprogramming        transcription factor in an animal cell having the first cell        fate to generate a cell that differentiates in response to        lineage-specific differentiating factors to a second cell fate        different from the first cell fate; and    -   submitting the cell that differentiates in response to        lineage-specific differentiating factors to conditions that        induce differentiation of the cell to generate a cell having the        second non-pluripotent cell fate.

In some embodiments, the development or growth of pluripotent cells islimited, e.g., by limiting the expression of, or contact of the cellsto, the reprogramming transcription factor(s), or by contacting the cellthat differentiates in response to lineage-specific differentiatingfactors with an inhibitor that inhibits the growth of pluripotent cells.In some embodiments, the inhibitor that inhibits the growth ofpluripotent cells is a JAK inhibitor.

In some embodiments, the cell that differentiates in response tolineage-specific differentiating factors does not significantly expressNanog.

In some embodiments, the method occurs in the absence of exogenous LIF.In some embodiments, the cell that differentiates in response tolineage-specific differentiating factors is not capable of forming ateratoma.

In some embodiments, the method does not comprise isolation or selectionof cells between the increasing and submitting steps.

In some embodiments, the cell that differentiates in response tolineage-specific differentiating factors is not an induced pluripotentstem cell.

In some embodiments, the animal cell having the first cell fate is afibroblast or a neural precursor cell. In some embodiments, the cellhaving the second non-pluripotent cell fate is a cardiomyocyte.

In some embodiments, the submitting step comprises contacting the cellthat differentiates in response to lineage-specific differentiatingfactors with BMP4, a calcium channel agonist (e.g., BayK 8644), a Gαsactivating agent, a cAMP analog, and/or a GSK-3 inhibitor.

In some embodiments, the cell having the second non-pluripotent cellfate has a neural cell fate. In some embodiments, the cell having thesecond non-pluripotent cell fate has a pancreatic cell fate.

In some embodiments, the reprogramming factors are selected from thegroup consisting of an Oct polypeptide, a Klf polypeptide, a Sox2polypeptide and a Myc polypeptide. In some embodiments, thereprogramming factors comprise an Oct polypeptide, a Klf polypeptide,and a Sox2 polypeptide. In some embodiments, the reprogramming factorscomprise an Oct polypeptide, a Klf polypeptide, and a Myc polypeptide.In some embodiments, the reprogramming factors comprise a Mycpolypeptide, a Klf polypeptide, and a Sox2 polypeptide. In someembodiments, the reprogramming factors comprise an Oct polypeptide, aSox2 polypeptide, and a Myc polypeptide. In some embodiments, thereprogramming factors comprise an Oct polypeptide, a Klf polypeptide, aSox2 polypeptide, and a Myc polypeptide. In some embodiments, thereprogramming factors comprise an Oct polypeptide and a Klf polypeptide.In some embodiments, the reprogramming factors comprise an Octpolypeptide and a Sox2 polypeptide. In some embodiments, thereprogramming factors comprise an Oct polypeptide and a Myc polypeptide.In some embodiments, the reprogramming factors comprise a Klfpolypeptide and a Sox2 polypeptide. In some embodiments, thereprogramming factors comprise a Klf polypeptide and a Myc polypeptide.In some embodiments, the reprogramming factors comprise a Sox2polypeptide and a Myc polypeptide.

In some embodiments, the time from initiation of the increasing step tothe generation of the cell having the second non-pluripotent cell fateis no more than 25, 24, 23, 22, or 21 days. In some embodiments, thetime from initiation of the increasing step to the generation of thecell having the second non-pluripotent cell fate is no more than 20, 19,18, 17, or 16 days. In some embodiments, the time from initiation of theincreasing step to the generation of the cell having the secondnon-pluripotent cell fate is no more than 15, 14, 13, 12, or 11 days. Insome embodiments, the time from initiation of the increasing step to thegeneration of the cell having the second non-pluripotent cell fate isbetween 8-22 days (i.e., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, or 22 days), between 9-22 days (i.e., 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, or 22 days), between 10-22 days (i.e., 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days), between 10-25 days(i.e., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25days), between 10-20 days (i.e., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,or 20 days), between 7-20 days (i.e., 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20 days), between 7-22 days (i.e., 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 days), or between 7-25days (i.e., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 days).

In some embodiments, the conditions that induce differentiation arechemically defined conditions.

In some embodiments, the method is conducted at least partly in vivo. Insome embodiments, the increasing step is performed in vivo. In someembodiments, the submitting step occurs by inducing the increasing stepin a cell in a cellular context in vivo such that endogenous signalscause the cell to have the second non-pluripotent cell fate.

In some embodiments, the method is conducted in vitro.

In another aspect, the present invention provides for methods ofdifferentiating an animal cell into a cardiomyocyte. In someembodiments, the method comprises contacting the animal cell with aGSK-3 inhibitor and/or a BMP protein (e.g., BMP4) under conditions togenerate a cardiomyocyte.

In some embodiments, the animal cell is a non-human animal cell. In someembodiments, the animal cell is a human cell.

In some embodiments, the cell is a pluripotent cell. In someembodiments, the cell is not a pluripotent cell.

In some embodiments, the method is conducted at least partly in vivo. Insome embodiments, the method is conducted in vitro.

In some embodiments, the conditions are chemically defined conditions.

In another aspect, the present invention provides for methods oftransdifferentiating an animal cell into a cardiomyocyte. In someembodiments, the method comprises:

-   -   introducing into a non-pluripotent animal cell having a first        non-pluripotent cell fate one or more polynucleotides encoding        one or more polypeptides selected from the group consisting of        an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a        Myc polypeptide; or contacting a non-pluripotent animal cell        having a first non-pluripotent cell fate with one or more        polypeptides selected from the group consisting of an Oct        polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc        polypeptide; and    -   contacting the cell with a GSK-3 inhibitor, a calcium channel        agonist (e.g., BayK 8644), a Gαs activating agent, a cAMP        analog, and/or a BMP protein (e.g., BMP4) under conditions to        generate the cardiomyocyte; thereby differentiating the cell        into the cardiomyocyte.

In some embodiments, the method further comprises contacting thenon-pluripotent animal cell with a JAK inhibitor. In some embodiments,the non-pluripotent animal cell is contacted with the JAK inhibitorprior to contacting the cell with the GSK-3 inhibitor, the calciumchannel agonist (e.g., BayK 8644), the Gαs activating agent, the cAMPanalog, and/or the BMP protein (e.g., BMP4). In some embodiments,contacting the non-pluripotent animal cell with the JAK inhibitorcomprises culturing the non-pluripotent animal cell in the presence ofthe JAK inhibitor. In some embodiments, the non-pluripotent animal cellis cultured in the presence of the JAK inhibitor for a period of aboutone to about nine days (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, or 9 days).

In yet another aspect, the present invention provides for methods oftransdifferentiating an animal cell into a neural progenitor cell. Insome embodiments, the method comprises:

-   -   introducing into a non-pluripotent animal cell having a first        non-pluripotent cell fate one or more polynucleotides encoding        one or more polypeptides selected from the group consisting of        an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a        Myc polypeptide; or contacting a non-pluripotent animal cell        having a first non-pluripotent cell fate with one or more        polypeptides selected from the group consisting of an Oct        polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc        polypeptide; and    -   contacting the cell with FGF2, FGF4, and EGF under conditions to        generate the neural progenitor cell; thereby differentiating the        cell into the neural progenitor cell.

In some embodiments, the method further comprises contacting thenon-pluripotent animal cell with a Wnt inhibitor, a JAK inhibitor, orboth a Wnt inhibitor and a JAK inhibitor. In some embodiments, thenon-pluripotent animal cell is contacted with the Wnt inhibitor, the JAKinhibitor, or both the Wnt inhibitor and the JAK inhibitor prior tocontacting the cell with FGF1, FGF4, and EGF.

In still another aspect, the present invention provides for methods oftransdifferentiating an animal cell into a retinal pigmented epitheliumcell. In some embodiments, the method comprises:

-   -   introducing into a non-pluripotent animal cell having a first        non-pluripotent cell fate one or more polynucleotides encoding        one or more polypeptides selected from the group consisting of        an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a        Myc polypeptide; or contacting a non-pluripotent animal cell        having a first non-pluripotent cell fate with one or more        polypeptides selected from the group consisting of an Oct        polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc        polypeptide;    -   contacting the cell with FGF2, FGF4, and EGF under conditions to        generate a neural progenitor cell; and    -   contacting the neural progenitor cell with a TGFβ inhibitor and        a GSK-3 inhibitor to generate the retinal pigmented epithelium        cell; thereby differentiating the cell into the retinal        pigmented epithelium cell.

In yet another aspect, the present invention provides for methods oftransdifferentiating an animal cell into a tyrosine hydroxylase(TH)-positive neuronal cell. In some embodiments, the method comprises:

-   -   introducing into a non-pluripotent animal cell having a first        non-pluripotent cell fate one or more polynucleotides encoding        one or more polypeptides selected from the group consisting of        an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a        Myc polypeptide; or contacting a non-pluripotent animal cell        having a first non-pluripotent cell fate with one or more        polypeptides selected from the group consisting of an Oct        polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc        polypeptide; and    -   contacting the cell with FGF8 and SHH under conditions to        generate the TH-positive neuronal cell; thereby differentiating        the cell into the TH-positive neuronal cell.

In still another aspect, the present invention provides for methods oftransdifferentiating an animal cell into a pancreatic lineage cell. Insome embodiments, the method comprises:

-   -   (1) introducing into a non-pluripotent animal cell having a        first non-pluripotent cell fate one or more polynucleotides        encoding one or more polypeptides selected from the group        consisting of an Oct polypeptide, a Klf polypeptide, a Sox2        polypeptide and a Myc polypeptide; or contacting a        non-pluripotent animal cell having a first non-pluripotent cell        fate with one or more polypeptides selected from the group        consisting of an Oct polypeptide, a Klf polypeptide, a Sox2        polypeptide and a Myc polypeptide; and    -   (2) under conditions to generate the pancreatic lineage cell,        -   (a) contacting the cell with a TGFβ/Activin/Nodal family            member and a JAK inhibitor;        -   (b) contacting the cell of step (2)(a) with a TGFβ/ALK5            receptor inhibitor, BMP4, bFGF, and RA; and        -   (c) contacting the cell of step (2)(b) with nicotinamide;            thereby differentiating the cell into the pancreatic lineage            cell.

In still another aspect, the present invention provides for methods oftransdifferentiating an animal cell into an induced definitive endoderm(iDE) cell. In some embodiments, the method comprises:

-   -   introducing into a non-pluripotent animal cell having a first        non-pluripotent cell fate one or more polynucleotides encoding        one or more polypeptides selected from the group consisting of        an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a        Myc polypeptide; or contacting a non-pluripotent animal cell        having a first non-pluripotent cell fate with one or more        polypeptides selected from the group consisting of an Oct        polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc        polypeptide; and    -   contacting the cell with a GSK-3 inhibitor, an HDAC inhibitor,        and a TGFβ/Activin/Nodal family member under conditions to        generate the iDE cell; thereby differentiating the cell into the        iDE cell.

In yet another aspect, the present invention provides for methods oftransdifferentiating an animal cell into a pancreatic beta cell. In someembodiments, the method comprises:

-   -   (1) introducing into a non-pluripotent animal cell having a        first non-pluripotent cell fate one or more polynucleotides        encoding one or more polypeptides selected from the group        consisting of an Oct polypeptide, a Klf polypeptide, a Sox2        polypeptide and a Myc polypeptide; or contacting a        non-pluripotent animal cell having a first non-pluripotent cell        fate with one or more polypeptides selected from the group        consisting of an Oct polypeptide, a Klf polypeptide, a Sox2        polypeptide and a Myc polypeptide;    -   (2) contacting the cell with a GSK-3 inhibitor, an HDAC        inhibitor, and a TGFβ/Activin/Nodal family member under        conditions to generate an iDE cell; and    -   (3) under conditions to generate the pancreatic beta cell,        -   (a) contacting the iDE cell with FGF7, RA, a Hedgehog            pathway inhibitor, a BMP inhibitor, and a TGFβ/ALK5 receptor            inhibitor;        -   (b) contacting the cell of step (3)(a) with EGF and a Notch            inhibitor; and        -   (c) contacting the cell of step (3)(b) with bFGF and            nicotinamide; thereby differentiating the cell into the            pancreatic beta cell.

In some embodiments, step (3) of the method further comprises contactingwith extendin 4. In some embodiments, step (3)(c) further comprisescontacting the cell of step (3)(b) with extendin 4.

DEFINITIONS

“Cell fate” as used herein is used as generally understood in the fieldof cell biology and refers to the ultimate differentiated state to whicha cell has become committed.

An “Oct polypeptide” refers to any of the naturally-occurring members ofOctamer family of transcription factors, or variants thereof thatmaintain transcription factor activity, similar (within at least 50%, atleast 60%, at least 70%, at least 80%, or at least 90% activity) ascompared to the closest related naturally occurring family member, orpolypeptides comprising at least the DNA-binding domain of the naturallyoccurring family member, and can further comprise a transcriptionalactivation domain. Exemplary Oct polypeptides include, Oct-1, Oct-2,Oct-3/4, Oct-6, Oct-7, Oct-8, Oct-9, and Oct-11. e.g., Oct3/4 (referredto herein as “Oct4”) which contains the POU domain, a 150 amino acidsequence conserved among Pit-1, Oct-1, Oct-2, and uric-86. See, Ryan, A.K. & Rosenfeld, M. G. Genes Dev. 11, 1207-1225 (1997). In someembodiments, variants have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95% or greater amino acid sequence identity across theirwhole sequence compared to a naturally occurring Oct polypeptide familymember such as those listed above or such as listed in Genbank accessionnumber NP_(—)002692.2 (human Oct4) or NP_(—)038661.1 (mouse Oct4). Octpolypeptides (e.g., Oct3/4) can be from human, mouse, rat, bovine,porcine, or other animals. Generally, the same species of protein willbe used with the species of cells being manipulated.

A “Klf polypeptide” refers to any of the naturally-occurring members ofthe family of Krüppel-like factors (Klfs), zinc-finger proteins thatcontain amino acid sequences similar to those of the Drosophilaembryonic pattern regulator Krüppel, or variants of thenaturally-occurring members that maintain transcription factor activitysimilar (within at least 50%, at least 60%, at least 70%, at least 80%,or at least 90% activity) as compared to the closest related naturallyoccurring family member, or polypeptides comprising at least theDNA-binding domain of the naturally occurring family member, and canfurther comprise a transcriptional activation domain. See, Dang, D. T.,Pevsner, J. & Yang, V. W. Cell Biol. 32, 1103-1121 (2000). Exemplary Klffamily members include, Klf1, Klf2, Klf3, Klf-4, Klf5, Klf6, Klf7, Klf8,Klf9, Klf10, Klf11, Klf12, Klf13, Klf14, Klf15, Klf16, and Klf17. Klf2and Klf-4 were found to be factors capable of generating iPS cells inmice, and related genes Klf1 and Klf5 did as well, although with reducedefficiency. See, Nakagawa, et al., Nature Biotechnology 26:101-106(2007). In some embodiments, variants have at least 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95% or greater amino acid sequenceidentity across their whole sequence compared to a naturally occurringKlf polypeptide family member such as those listed above or such aslisted in Genbank accession number CAX16088 (mouse Klf4) or CAX14962(human Klf4). Klf polypeptides (e.g., Klf1, Klf4, and Klf5) can be fromhuman, mouse, rat, bovine, porcine, or other animals. Generally, thesame species of protein will be used with the species of cells beingmanipulated. To the extent a Klf polypeptide is described herein, it canbe replaced with an estrogen-related receptor beta (Essrb) polypeptide.Thus, it is intended that for each Klf polypeptide embodiment describedherein, a corresponding embodiment using Essrb in the place of a Klf4polypeptide is equally described.

A “Myc polypeptide” refers any of the naturally-occurring members of theMyc family (see, e.g., Adhikary, S. & Eilers, M. Nat. Rev. Mol. CellBiol. 6:635-645 (2005)), or variants thereof that maintain transcriptionfactor activity similar (within at least 50%, at least 60%, at least70%, at least 80%, or at least 90% activity) as compared to the closestrelated naturally occurring family member, or polypeptides comprising atleast the DNA-binding domain of the naturally occurring family member,and can further comprise a transcriptional activation domain. ExemplaryMyc polypeptides include, e.g., c-Myc, N-Myc and L-Myc. In someembodiments, variants have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95% or greater amino acid sequence identity across theirwhole sequence compared to a naturally occurring Myc polypeptide familymember, such as those listed above or such as listed in Genbankaccession number CAA25015 (human Myc). Myc polypeptides (e.g., c-Myc)can be from human, mouse, rat, bovine, porcine, or other animals.Generally, the same species of protein will be used with the species ofcells being manipulated.

A “Sox polypeptide” refers to any of the naturally-occurring members ofthe SRY-related HMG-box (Sox) transcription factors, characterized bythe presence of the high-mobility group (HMG) domain, or variantsthereof that maintain transcription factor activity similar (within atleast 50%, at least 60%, at least 70%, at least 80%, or at least 90%activity) as compared to the closest related naturally occurring familymember, or polypeptides comprising at least the DNA-binding domain ofthe naturally occurring family member, and can further comprise atranscriptional activation domain. See, e.g., Dang, D. T., et al., Int.J. Biochem. Cell Biol. 32:1103-1121 (2000). Exemplary Sox polypeptidesinclude, e.g., Sox1, Sox-2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9,Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17, Sox18, Sox-21, andSox30. Sox1 has been shown to yield iPS cells with a similar efficiencyas Sox2, and genes Sox3, Sox15, and Sox18 have also been shown togenerate iPS cells, although with somewhat less efficiency than Sox2.See, Nakagawa, et al., Nature Biotechnology 26:101-106 (2007). In someembodiments, variants have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95% or greater amino acid sequence identity across theirwhole sequence compared to a naturally occurring Sox polypeptide familymember such as those listed above or such as listed in Genbank accessionnumber CAA83435 (human Sox2). Sox polypeptides (e.g., Sox1, Sox2, Sox3,Sox15, or Sox18) can be from human, mouse, rat, bovine, porcine, orother animals. Generally, the same species of protein will be used withthe species of cells being manipulated.

A “recombinant” polynucleotide is a polynucleotide that is not in itsnative state, e.g., the polynucleotide comprises a nucleotide sequencenot found in nature, or the polynucleotide is in a context other thanthat in which it is naturally found, e.g., separated from nucleotidesequences with which it typically is in proximity in nature, or adjacent(or contiguous with) nucleotide sequences with which it typically is notin proximity. For example, the sequence at issue can be cloned into avector, or otherwise recombined with one or more additional nucleicacid.

“Expression cassette” refers to a polynucleotide comprising a promoteror other regulatory sequence operably linked to a sequence encoding aprotein.

The terms “promoter” and “expression control sequence” are used hereinto refer to an array of nucleic acid control sequences that directtranscription of a nucleic acid. As used herein, a promoter includesnecessary nucleic acid sequences near the start site of transcription,such as, in the case of a polymerase II type promoter, a TATA element. Apromoter also optionally includes distal enhancer or repressor elements,which can be located as much as several thousand base pairs from thestart site of transcription. Promoters include constitutive andinducible promoters. A “constitutive” promoter is a promoter that isactive under most environmental and developmental conditions. An“inducible” promoter is a promoter that is active under environmental ordevelopmental regulation. The term “operably linked” refers to afunctional linkage between a nucleic acid expression control sequence(such as a promoter, or array of transcription factor binding sites) anda second nucleic acid sequence, wherein the expression control sequencedirects transcription of the nucleic acid corresponding to the secondsequence.

A “heterologous sequence” or a “heterologous nucleic acid”, as usedherein, is one that originates from a source foreign to the particularhost cell, or, if from the same source, is modified from its originalform. Thus, a heterologous expression cassette in a cell is anexpression cassette that is not endogenous to the particular host cell,for example by being linked to nucleotide sequences from an expressionvector rather than chromosomal DNA, being linked to a heterologouspromoter, being linked to a reporter gene, etc.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein to refer to deoxyribonucleotides or ribonucleotides and polymersthereof in either single- or double-stranded form. The term encompassesnucleic acids containing known nucleotide analogs or modified backboneresidues or linkages, which are synthetic, naturally occurring, andnon-naturally occurring, which have similar binding properties as thereference nucleic acid, and which are metabolized in a manner similar tothe reference nucleotides. Examples of such analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleicacids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences, as well as thesequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Naturally encoded amino acids arethe 20 common amino acids (alanine, arginine, asparagine, aspartic acid,cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,leucine, lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine) and pyrrolysine and selenocysteine.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and

8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

“Inhibitors,” “activators,” and “modulators” of expression or ofactivity are used to refer to inhibitory, activating, or modulatingmolecules, respectively, identified using in vitro and in vivo assaysfor expression or activity of a described target protein, e.g., ligands,agonists, antagonists, and their homologs and mimetics. The term“modulator” includes inhibitors and activators. Inhibitors are agentsthat, e.g., inhibit expression or bind to, partially or totally blockstimulation or protease inhibitor activity, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate the activity ofthe described target protein, e.g., antagonists. Activators are agentsthat, e.g., induce or activate the expression of a described targetprotein or bind to, stimulate, increase, open, activate, facilitate,enhance activation or protease inhibitor activity, sensitize or upregulate the activity of described target protein (or encodingpolynucleotide), e.g., agonists. Modulators include naturally occurringand synthetic ligands, antagonists and agonists (e.g., small chemicalmolecules, antibodies and the like that function as either agonists orantagonists). Such assays for inhibitors and activators include, e.g.,applying putative modulator compounds to cells expressing the describedtarget protein and then determining the functional effects on thedescribed target protein activity, as described above. Samples or assayscomprising described target protein that are treated with a potentialactivator, inhibitor, or modulator are compared to control sampleswithout the inhibitor, activator, or modulator to examine the extent ofeffect. Control samples (untreated with modulators) are assigned arelative activity value of 100%. Inhibition of a described targetprotein is achieved when the activity value relative to the control isabout 80%, optionally 50% or 25, 10%, 5% or 1%. Activation of thedescribed target protein is achieved when the activity value relative tothe control is 110%, optionally 150%, optionally 200, 300%, 400%, 500%,or 1000-3000% or more higher.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Development of robust conditions for direct reprogramming offibroblasts to a mature cardiac fate. (A) X-gal staining ofNebulette-LacZ⁺ colonies arising seven days after three-factortransduction. (B) An outline of the final direct cardiac reprogrammingconditions, with the onset of spontaneous contraction indicated. (C, D)Immunostaining of mid- and late-stage cardiac lineage markers on days11-15 and 18-21, respectively. Scale bars in (A), (C), and (D) are 100μm.

FIG. 2: Gauging reprogramming efficiency and success by incidence ofbeating and marker expression. (A, B) Changes in contracting colonynumber and percentage, respectively, upon culture treatment with BMP4alone or in combination with the JAK inhibitor JI1. (C) Whole-wellimaging (96-well plate format) depicting the relative expression of cTnTand Nanog on day 18. (D) FACS counts of cells positive for the indicatedmarkers at three different timepoints. Scale bar in (C) is 900 μm.

FIG. 3: Calcium flux and electrophysiological characterization ofcontracting cardiomyocytes. (A) Calcium transients recorded inspontaneously contracting colonies, before and after isoproterenol (Iso)and carbachol (CCh) treatment at the indicated concentrations.Quantification of the effect of treatment on transient frequency (B) anddecay rate (C). (D) Spontaneous action potentials recorded from anindividual cardiomyocyte at 20-22° C. The waveform of a single actionpotential is shown in the lower inset using an expanded time scale.Dashed lines indicate resting potential (−62 mV) and 0 mV, respectively.Colony numbers in a and b correspond to incidence per 100,000 MEFsplated and were recorded on day 18; error bars indicate s.e.m, n=6. Thedemarcations ** and *** indicate p values of >0.01 and >0.001,respectively. (E) Immunostaining of individually contracting cellsisolated from a large colony arising from fibroblasts treated withcardiac reprogramming conditions.

FIG. 4: Development of pluripotency is detrimental to cardiogenesis. (A)ChIP assays examining epigenetic modifications at four relevant lociduring reprogramming, as indicated by changes in K4 and K27trimethylation levels of histone H3. CorAT denotes commerciallyavailable pure cardiomyocytes. (B) Effect of conditions promoting iPSCgeneration on cardiac reprogramming. Schematic depiction ofmodifications to conditions (left) with corresponding relativeefficiencies (right). (C) Testing of our protocol (and portions thereof,indicated by grey arrows) on mESCs to ascertain potential cardiogeniceffects on pluripotent cells. (D) Differential effects of prolongedtransgene expression on beating vs. total colony number.

FIG. 5: Direct cardiac reprogramming represents a parallel process thatoccurs in the absence of iPSC generation. Time course analysis ofcardiac lineage (A) and pluripotency (B) marker expression byquantitative RT-PCR. (C) Phase-contrast and FITC-channel imagesdepicting cardiac colony formation and maturation in Nanog-GFP reporterMEFs. Spontaneous contraction was first detected on day 12 in theencircled portion of the colony. Robust early activation of theNanog-GFP marker using standard reprogramming methods is provided as apositive control. Error bars in (A) and (B) indicate standard deviation,n=3. Scale bars in (C) are 100 μm.

FIG. 6: A model for direct reprogramming to alternative fates. A briefburst of reprogramming factor overexpression leads to the formation ofvarious highly transient and epigenetically unstable (i.e., lessrestricted, more naïve) intermediates. These cells then spontaneously“relax” back into more stable state(s). Due to the very complex natureof complete dedifferentiation, the generation of iPSCs is not a likelyoutcome of this process, and requires continued overexpression ofexogenous pluripotency factors. Conversely, a direct switch tomultipotent lineage progenitors—or even terminally differentiatedcells—represents the path of least resistance, especially under cultureconditions that (a) allow and/or promote their genesis and proliferationand (b) simultaneously inhibit establishment of pluripotency (JAK/STATinhibition).

FIG. 7: Transdifferentiation by transient expression of the conventionalfour reprogramming factors generates functional neural stem/progenitorcells. (A) Scheme for the transdifferentiation of Dox-induciblesecondary MEF cells into neural stem/progenitor cells (NPCs). Durationof dox (4 μg/ml) treatment is for the indicated number of days.Different media were added sequentially as described in Methods. (B)Pax6 immunostaining on day 13 of colonies arising from the indicateddurations of dox treatment. 8 μg/ml dox was used for the three-daytreatment. (C) Number of PLZF expressing colonies generated withdifferent durations of dox treatment, as analyzed on day 13. (D)Immunostaining of colonies on day 13 with various neural or neuronalmarkers. (E) Immunostaining of spontaneously differentiated cells fromisolated colonies on day 13 with various mature neuronal or glialmarkers. All scale bars represent 100 μm.

FIG. 8: Direct reprogramming is a highly efficient method of derivingpure neural stem/progenitor cells. (A) Experimental overview and cartoonrepresentation of results from panels B, D, and F. Cells after nine daysof differentiation from iPSCs, or after 13 days of transdifferentiation,show the indicated marker expression profiles. (B) Day 9 immunostainingof cells differentiated from iPSCs, or transdifferentiated NPCs on day13, with antibodies against the indicated markers. Pax6 and Sox1demarcate early neuroectoderm. Sox17 is indicative of endoderm. Texpression is early mesodermal. All scale bars represent 100 μm. (C-F)qRT-PCR analysis of the indicated markers' expression in cells harvestedat multiple timepoints during the direct reprogramming process (C,D) anddifferentiation from iPSCs (E,F). Pluripotency genes (C,E) andlineage-specific genes (D,F) are shown in separate graphs. All valuesare relative to expression in iPSCs.

FIG. 9: Fate choice is dictated early during the transdifferentiationprocess by different environmental cues. (A) Schematic of experimentsinvolving differential exposure to LIF-containing medium (RepM-Pluri)from day 4 onwards for the indicated number of days. Dox treatment wasfor five days, beginning on day 0 (D0). (B) PLZF-positive colony numberexpressed as a percentage of the total from each indicated sample on day9. (C,D) Quantitative analysis of mRNA levels of indicated marker genesin each sample (harvested on day 9). All values are relative toexpression in iPSCs. Pax6 immunostaining (E) and total colony number (F)of TTFs transdifferentiated in the presence or absence of JAK inhibitor.

FIG. 10: A model for direct reprogramming of MEFs to neuralstem/progenitor cells. By adding neural medium to four factor-inducedintermediate cells comprising various epigenetic states, a fate switchto neural stem/progenitor cells (NPCs) can be achieved. Alternatively,iPSCs can be generated by prolonged expression of the four factors withconcomitant incubation in ESC medium. Cells belonging to other lineagescould likely also be isolated, depending on the type of medium used.

FIG. 11: Flow cytometric analysis of the transdifferentiated NSCs. (A)Forward- and side-scatter FACS plots of day 7 and day 12 samples. In theday 12 sample, cells were divided into two subsets based on grossdistribution. Subset 2 is not as prominent on day 7 as on day 12.Subsets 1 and 2 most likely represent the populations of unchangedfibroblasts and colony-forming transdifferentiated cells, respectively.(B) Histograms of cells from day 12 stained with anti-SSEA1, Prominin-1and A2B5 antibodies. Differently gated (total, subset 1 or subset 2)cells were analyzed separately. (C) Histograms of cells from day 7 andday 12 stained with PSA-NCAM antibody.

FIG. 12: Flow cytometric analysis of cells temporarily exposed to LIF.(A) Schematic of experiments with differential exposure toLIF-containing medium (RepM-Pluri) from day 3 onwards for 0, 2, 4, and 8days, respectively. Dox treatment was for five days, beginning on day 0(D0). (B) Percentage of SSEA1-expressing cells on days 9 and 11. (C)Histograms of SSEA1-expressing cells, as analyzed by flow cytometry.

FIG. 13: Quantitation of sequential gene expression duringtransdifferentiation. The experimental setup is the same as in FIG. 9A,with samples harvested at the indicated time points. Pluripotency genes(A) and lineage-specific marker genes (B) are shown in separate graphs.All values are relative to expression levels in iPSCs. Dox treatment wasfor five days, beginning on day 0.

FIG. 14: Direct reprogramming of human fibroblasts to definitiveendoderm (iDE) by Oct4 and a unique endodermal inducing condition. (A)shows iDEs positive for both SOX17 and FOXA2, typical markers fordefinitive endoderm. (B) Bisulfate sequencing analysis shows that SOX17and FOXA2 promoters are largely demethylated. (C) Expression analysis ofpluripotency genes and definitive endoderm marker genes in iDEs.

FIG. 15: Direct reprogramming of human fibroblasts to pancreatic lineagecells, such as pancreatic β-like cells. (A) Experimental overview andcartoon representation of results. (B) Immunostaining of iDEs withantibodies against the indicated markers. (B) Real-time PCR analysis ofiDE induced pancreatic β-like cells. Expression of islet specificmarkers genes, including PDX1, NKX6.1, MAFA, GLUT2, GLUCOKINASE andINSULIN, were analyzed. (D) Analysis of C-Peptide released frompancreatic β-like cells derived from iDEs following in vitro glucosestimulation.

FIG. 16: Transdifferentiation of Dox-inducible secondary mousefibroblast to pancreatic β-like cells. (A) Schematic overview of theinduction protocol. (B) Immunostaining of the induced pancreatic β-likecells with antibody specific to C-peptide.

FIG. 17: Nkx2.5 GFP Targeting Vector.

FIG. 18: Nkx2.5-GFP Cardiomyocytes Express Low Levels of GFP.

FIG. 19: Transcript Expression Levels in Cardiomyocytes.

FIG. 20: MLC-2v-GFP Cells Express GFP in Beating Patches.

FIG. 21: A Permissive Percentage of MLC-2v-GFP Cells Differentiate IntoCardiomyocytes.

FIG. 22: Pintool Technology Delivers Nanoliters of Compound.

FIG. 23: A Potential Hit From MLC-2v Screen Displays Strong Fluorescencein GFP Channel.

FIG. 24: Schematic for the Stepwise Differentiation of Cardiomyocytes.

FIG. 25: Sufficient ESC Colony Size is Required for Survival in CDM.

FIG. 26: Molecular Structure of GSK-3 inhibitor,6-bromoindirubin-3′-oxime (BIO).

FIG. 27: BIO Induces Expression of αMHC and GATA-4.

FIG. 28: BMP-4 Induces Expression of αMHC and GATA-4.

FIG. 29: RT-PCR Time Course Analysis of Differentiation.

FIG. 30: Differentiating Cells Express Intermediate Markers of CardiacDifferentiation.

FIG. 31: Beating Cells Express Markers of Mature Cardiomyocytes.

FIG. 32: Beating Cells Express Markers of Mature Cardiomyocytes.

FIG. 33: Molecular Structure of BayK 8644.

FIG. 34: Percentage of Beating Colonies.

FIG. 35: FACS Analysis of Mesodermal Markers. Cells were treated withthe small molecule BayK 8644 (A), isoprotenolol (B), dibutyrl cAMP (C),or propanolol (D) and Brachyury expression was measured.

FIG. 36: QRT-pCR Analysis of Markers. Normalized fold expression ofmarkers was measured for cells treated with BayK 8644, dibutyrl cAMP,isoprotenolol, or propanolol.

FIG. 37: Molecular Structure of TGF-beta Inhibitor, A83-01.

FIG. 38: Percentage Beating Colonies Observed After Treatment in Days5-7.

FIG. 39: QRT-PCR After Treatments in Days 5-7.

DETAILED DESCRIPTION I. Introduction

Methods and compositions for efficiently transdifferentiating a cellfrom a first cell fate to a second fate are provided. For example, acell first differentiates into a first cell fate. During thetransdifferentiation, the cell is changed from a first cell fate into adifferent cell fate. In some embodiments, the cell transdifferentiatesfrom one cell type (e.g., cells of ectoderm, mesoderm, or endoderm) to adifferent cell type. In some embodiments, an ectoderm celltransdifferentiates into an endoderm cell. In some embodiments, anectoderm cell transdifferentiates into a mesoderm cell. In someembodiments, an endoderm cell transdifferentiates into an ectoderm cell.In some embodiments, an endoderm cell transdifferentiates into amesoderm cell. In some embodiments, a mesoderm cell transdifferentiatesinto an endoderm cell. In some embodiments, a mesoderm celltransdifferentiates into an ectoderm cell. In some embodiments, themethod of transdifferentiation comprises at least two steps:

-   -   (1) submitting an animal cell having a first cell fate to        conditions to generate a cell (i.e., a less differentiated cell)        that is capable of differentiating into a second cell fate; and    -   (2) submitting the less differentiated cell to conditions to        differentiate the cell into a cell having the second cell fate.

For example, the inventors have discovered that it is possible to treata differentiated cell (i.e., mouse embryonic fibroblasts (MEFs) orneural precursor cells) in culture to generate a less differentiatedcell that can be subsequently differentiated into any of cardiac cells(e.g., cardiomyoctes), neuronal cells (e.g., when MEFs are initiallyused), or pancreatic cells. Thus, it is believed that the method isgenerally applicable for transdifferentiating one cell type to anotherwithout generating a pluripotent intermediate, or at least withoutgeneration of an induced pluripotent (iPS) cell. For example, theinventors have found that the transdifferentiation method does notrequire the inclusion of LIF, which is typically required formaintenance of iPS cells. Moreover, the transitory less differentiatedcells generated express little or no Nanog and thus can be distinguishedfrom iPS cells by substantial lack of expression of Nanog. To the extentNanog or Oct4 expression was observed in beating colonies whenconditions to generate cardio cells were used, only very large coloniessometimes contained both a beating patch and some very weak Nanog orOct4 expression, but these areas never overlapped. Finally, the timingof generation of the new cells (e.g., the cardiomyocytes) measured fromthe start of reprogramming is considerably faster than would be expectedif the cells were generated via an induced pluripotent stem cellintermediate.

II. Generation of Undifferentiated Cells from Cells Having a FirstCommitted Cell Fate

The inventors have found that applying partial, but not complete, iPSreprogramming conditions to differentiated cells can result in cellsthat are capable of differentiating into a second cell fate, i.e., to adifferent cell lineage than the first cell fate. As explained below, theinventors have found that induction of retrovirally-delivered three(Oct4, Klf4, Sox2) or four (Oct4, Klf4, Sox2, c-Myc) “Yamanaka factors”in serum but the absence of LIF (i.e., a factor generally necessary forsignificant iPS cell production) was sufficient to generate cells thatcould subsequently and directly be induced to other lineages. While thespecific conditions used by the inventors can be used, it is believedthat other iPS reprogramming conditions can also be modified such thatsubstantially no iPS cells are generated and yet cells capable ofaltered differentiation are produced. To date, a large number ofdifferent methods and protocols have been established for inducingnon-pluripotent mammalian cells into iPS cells. iPS cells are similar toESCs in morphology, proliferation, and pluripotency, judged by teratomaformation and chimaera contribution. Reprogramming protocols that can bemodified as described herein are believed to include those involvingintroduction of one or more reprogramming transcription factors selectedfrom an Oct polypeptide (including but not limited to Oct 3/4), a Soxpolypeptide (including but not limited to Sox2), a Klf polypeptide(including but not limited to Klf4) and/or a Myc polypeptide (includingbut not limited to c-Myc). The reprogramming factors can be introducedinto the cells, for example, by expression from a recombinant expressioncassette that has been introduced into the target cell, or by incubatingthe cells in the presence of exogenous reprogramming transcriptionfactor polypeptides such that the polypeptides enter the cell.

Studies have shown that retroviral transduction of mouse fibroblastswith four transcription factors that are highly expressed in ESCs(Oct-3/4, Sox2, KLF4 and c-Myc) generate induced pluripotent stem (iPS)cells in combination with LIF. See, Takahashi, K. & Yamanaka, S. Cell126, 663-676 (2006); Okita, K., Ichisaka, T. & Yamanaka, S, Nature 448,313-317 (2007); Wernig, M. et al. Nature 448, 318-324 (2007); Maherali,N. et al. Cell Stem Cell 1, 55-70 (2007); Meissner, A., Wernig, M. &Jaenisch, R. Nature Biotechnol. 25, 1177-1181 (2007); Takahashi, K. etal. Cell 131, 861-872 (2007); Yu, J. et al. Science 318, 1917-1920(2007); Nakagawa, M. et al. Nature Biotechnol. 26, 101-106 (2007);Wernig, M., Meissner, A., Cassady, J. P. & Jaenisch, R. Cell Stem Cell.2, 10-12 (2008). Such methods can be altered to lack LIF, or otherwisenot generate iPS cells (including but not limited to the specificmethods for blocking iPS cell generation described herein), and yet becapable of re-differentiation of cells to a second cell fate. As analternative to omitting LIF, or in combination, to reduce the possiblecontamination of pluripotent cells, some chemical compounds that inhibitthe growth of pluripotent cells can be included in the cell culturemedia or otherwise contacted to the target cells. Exemplary chemicalcompounds include, but are not limited to, PI3K inhibitors, CDK2inhibitors, Wnt inhibitors, or a combination thereof.

In embodiments involving expression in the cell or contact to the cellof one or more reprogramming transcription factors, the methods caninvolve limiting expression or exposure of the cells to thereprogramming transcription factors. For example, in some embodiments,expression of the factors is induced (e.g., by an inducible promoter)for a limited time that is insufficient to produce iPS cells. Forexample, in some embodiments, the transcription factor(s) are inducedfor between 1-9, 2-8, 3-6 days. Expression can subsequently be turnedoff by changing the media or condition of the cells to remove theinducer. Similarly, in embodiments in which the cells are exposed toprotein transcription factors (discussed more below), the cells arecontacted to the protein for a limited time, thereby reducing orpreventing development of iPS cells.

This invention employs routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)). In some embodiments, expression cassettesfor expression of one or more reprogramming transcription factor isintroduced into a cell.

In some embodiments, the species of cell and protein to be expressed isthe same. For example, if a mouse cell is used, a mouse ortholog oractive protein variant thereof is introduced into the cell. If a humancell is used, a human ortholog protein or active variant thereof isintroduced into the cell. Alternatively, in some embodiments, thespecies of cell and protein are not matched.

It will be appreciated that where two or more proteins are to beexpressed in a cell (e.g., two or more polypeptides selected from thegroup consisting of an Oct polypeptide, a Klf polypeptide, a Sox2polypeptide, and a Myc polypeptide), one or multiple expressioncassettes can be used. For example, where one expression cassetteexpresses multiple polypeptides, a polycistronic expression cassette canbe used.

Any type of vector can be used to introduce an expression cassette ofthe invention into a cell. Exemplary vectors include but are not limitedto plasmids and viral vectors. Exemplary viral vectors include, e.g.,adenoviral vectors, AAV vectors, and retroviral (e.g., lentiviral)vectors.

Suitable methods for nucleic acid delivery for transformation of a cell,a tissue or an organism for use with the current invention are believedto include virtually any method by which a nucleic acid (e.g., DNA) canbe introduced into a cell, a tissue or an organism, as described hereinor as would be known to one of ordinary skill in the art (e.g.,Stadtfeld and Hochedlinger, Nature Methods 6(5):329-330 (2009); Yusa etal., Nat. Methods 6:363-369 (2009); Woltjen et al., Nature 458, 766-770(9 Apr. 2009)). Such methods include, but are not limited to, directdelivery of DNA such as by ex vivo transfection (Wilson et al., Science,244:1344-1346, 1989, Nabel and Baltimore, Nature 326:711-713, 1987),optionally with Fugene6 (Roche) or Lipofectamine (Invitrogen), byinjection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448,5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, eachincorporated herein by reference), including microinjection (Harland andWeintraub, J. Cell Biol., 101:1094-1099, 1985; U.S. Pat. No. 5,789,215,incorporated herein by reference); by electroporation (U.S. Pat. No.5,384,253, incorporated herein by reference; Tur-Kaspa et al., Mol. CellBiol., 6:716-718, 1986; Potter et al., Proc. Nat'l Acad. Sci. USA,81:7161-7165, 1984); by calcium phosphate precipitation (Graham and VanDer Eb, Virology, 52:456-467, 1973; Chen and Okayama, Mol. Cell Biol.,7(8):2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10:689-695, 1990);by using DEAE-dextran followed by polyethylene glycol (Gopal, Mol. CellBiol., 5:1188-1190, 1985); by direct sonic loading (Fechheimer et al.,Proc. Nat'l Acad. Sci. USA, 84:8463-8467, 1987); by liposome mediatedtransfection (Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190,1982; Fraley et al., Proc. Nat'l Acad. Sci. USA, 76:3348-3352, 1979;Nicolau et al., Methods Enzymol., 149:157-176, 1987; Wong et al., Gene,10:87-94, 1980; Kaneda et al., Science, 243:375-378, 1989; Kato et al.,J Biol. Chem., 266:3361-3364, 1991) and receptor-mediated transfection(Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, J. Biol. Chem.,262:4429-4432, 1987); and any combination of such methods, each of whichis incorporated herein by reference.

To address the safety issues that arise from target cell genomesharboring integrated exogenous sequences, a number of modified geneticprotocols have been further developed and can be modified according tothe present invention. These protocols produce iPS cells withpotentially reduced risks, and include non-integrating adenoviruses todeliver reprogramming genes (Stadtfeld, M., et al. (2008) Science 322,945-949), transient transfection of reprogramming plasmids (Okita, K.,et al. (2008) Science 322, 949-953), piggyBac transposition systems(Woltjen, K., et al. (2009). Nature 458, 766-770, Yusa et al. (2009)Nat. Methods 6:363-369, Kaji, K., et al. (2009) Nature 458, 771-775),Cre-excisable viruses (Soldner, F., et al. (2009) Cell 136, 964-977),and oriP/EBNA1-based episomal expression system (Yu, J., et al. (2009)Science DOI: 10.1126). Such methods can be modified for example asdescribed herein.

In some embodiments, reprogramming can involve culturing target cells inthe presence of one or more proteins under conditions to allow forintroduction of the proteins into the cell. See, e.g., Zhou H et al.,Cell Stem Cell. 2009 May 8; 4(5):381-4; WO/2009/117439. One canintroduce an exogenous polypeptide (i.e., a protein provided fromoutside the cell and/or that is not produced by the cell) into the cellby a number of different methods that do not involve introduction of apolynucleotide encoding the polypeptide. In some embodiments, theexogenous proteins comprise the transcription factor polypeptide ofinterest linked (e.g., linked as a fusion protein or otherwisecovalently or non-covalently linked) to a polypeptide that enhances theability of the transcription factor to enter the cell (and in someembodiments the cell nucleus).

Examples of polypeptide sequences that enhance transport acrossmembranes include, but are not limited to, the Drosophila homeoproteinantennapedia transcription protein (AntHD) (Joliot et al., New Biol. 3:1121-34, 1991; Joliot et al., Proc. Natl. Acad. Sci. USA, 88: 1864-8,1991; Le Roux et al., Proc. Natl. Acad. Sci. USA, 90: 9120-4, 1993), theherpes simplex virus structural protein VP22 (Elliott and O'Hare, Cell88: 223-33, 1997); the HIV-1 transcriptional activator TAT protein(Green and Loewenstein, Cell 55: 1179-1188, 1988; Frankel and Pabo, Cell55: 1 289-1193, 1988); Kaposi FGF signal sequence (kFGF); proteintransduction domain-4 (PTD4); Penetratin, M918, Transportan-10; anuclear localization sequence, a PEP-I peptide; an amphipathic peptide(e.g., an MPG peptide); delivery enhancing transporters such asdescribed in U.S. Pat. No. 6,730,293 (including but not limited to anpeptide sequence comprising at least 5-25 or more contiguous argininesor 5-25 or more arginines in a contiguous set of 30, 40, or 50 aminoacids; including but not limited to an peptide having sufficient, e.g.,at least 5, guanidino or amidino moieties); and commercially availablePenetratin™ 1 peptide, and the Diatos Peptide Vectors (“DPVs”) of theVectocell® platform available from Daitos S.A. of Paris, France. Seealso, WO/2005/084158 and WO/2007/123667 and additional transportersdescribed therein. Not only can these proteins pass through the plasmamembrane but the attachment of other proteins, such as the transcriptionfactors described herein, is sufficient to stimulate the cellular uptakeof these complexes. A number of polypeptides capable of mediatingintroduction of associated molecules into a cell have been describedpreviously and can be adapted to the present invention. See, e.g.,Langel (2002) Cell Penetrating Peptides CRC Press, Pharmacology andToxicology Series.

Exemplary polypeptide sequences that enhance transport across membranesinclude:

VP22: G S P P T A P T R S K T P A Q G L A R K L H F ST A P P N P D A P W T P R V A G F N K R V F R FS P Q T A R R A T T T R I; kFGF:A G S G G A A V A L L P A V L L A L L A P G G E F A; PTD4:A G S G G Y A R A A A R Q A R A G G E F A; PENETRATIN:R Q I K I W F Q G R R M K W K K; TAT: Y G R K K R R Q R R R; M918:M V T V L F R R L R I R R A C G P P R V R V; TRANSPORTAN-10:A G Y L L G K I G L K A L A A L A K K I L.

In some embodiments, the polypeptide that enhances transport acrossmembranes is a peptide sequence comprising at least 5 or more contiguousor non-contiguous arginines (e.g., a 8-arginine peptide). In someembodiments, the polypeptide that enhances transport across membranes isa peptide sequence comprising at least 7 or more contiguous ornon-contiguous arginines. For example, the polypeptide that enhancestransport across membranes is a peptide sequence comprising 11contiguous arginines, e.g., ESGGGGSPGRRRRRRRRRRR. As noted above, thearginines in the transport enhancing sequence need not all becontiguous. In some embodiments, the polyarginine (e.g., the contiguousor non-contiguous) region is at least 5, 8, 10, 12, 15, 20, or moreamino acids long and has at least, e.g., 40%, 50%, 60%, 70%, 80%, 90%,or more arginines.

An exogenous polypeptide can be introduced into cells by traditionalmethods such as lipofection, electroporation, calcium phosphateprecipitation, particle bombardment and/or microinjection, or can beintroduced into cells by a protein delivery agent. For example, theexogenous polypeptide can be introduced into cells by covalently ornoncovalently attached lipids, e.g., by a covalently attached myristoylgroup. Lipids used for lipofection are optionally excluded from cellulardelivery modules in some embodiments. In some embodiments, thetranscription factor polypeptides described herein are exogenouslyintroduced as part of a liposome, or lipid cocktail (such ascommercially available Fugene®6 and Lipofectamine™). In anotheralternative, the transcription factor proteins can be microinjected orotherwise directly introduced into the target cell. In some embodiments,the transcription factor polypeptides are delivered into cells usingProfect protein delivery reagents, e.g., Profect-P1 and Profect-P2(Targeting Systems, El Cajon, Calif.), or using Pro-Ject® transfectionreagents (Pierce, Rockford Ill., USA). In some embodiments, thetranscription factor polypeptides are delivered into cells using asingle-wall nano tube (SWNT).

As discussed in the Examples of WO/2009/117439, incubation of cells withthe transcription factor polypeptides of the invention for extendedperiods can be toxic to the cells. Therefore, in some embodiments of theinvention, the non-pluripotent mammalian cells are intermittentlyincubated with one or more of an Oct polypeptide (including but notlimited to Oct 3/4), a Sox polypeptide (including but not limited toSox2), a Klf polypeptide (including but not limited to Klf4) and/or aMyc polypeptide (including but not limited to c-Myc) with interveningperiods of incubation of the cells in the absence of the one or morepolypeptides. In some embodiments, the cycle of incubation with andwithout the polypeptides can be repeated for 2, 3, 4, 5, 6, or moretimes but is not performed for sufficient lengths of time (i.e., theincubations with and without proteins) to achieve the development ofpluripotent cells.

A variety of agents (e.g., HDAC inhibitor, TGFβ receptor/ALK5 inhibitor,MEK/ERK pathway inhibitor, and/or Rho GTPase/ROCK inhibitor, etc.) canbe contacted to non-pluripotent cells either prior to, simultaneouswith, or after delivery of, programming transcription factors (forexample, delivered via expression cassette or as proteins). Exemplarysmall molecules that can be used are described in, e.g., WO/2009/117439.For convenience, the day the reprogramming factors are delivered isdesignated “day 1”. In some embodiments, the inhibitors are contacted tocells in aggregate (i.e., as a “cocktail”) at about days 3-7 andcontinued for 7-14 days. Alternatively, in some embodiments, thecocktail is contacted to the cells at day 0 (i.e., a day before thepreprogramming factors) and incubated for about 14-30 days.

The cell into which a protein of interest is introduced can be amammalian cell. The cells can be human or non-human (e.g., primate, rat,mouse, rabbit, bovine, dog, cat, pig, etc.). The cell can be, e.g., inculture or in a tissue, fluid, etc. and/or from or in an organism.

Optionally, or in addition, small molecules can “complement” or replacewhat is generally otherwise understood as a necessary expression of oneof these proteins to result in pluripotent cells. By contacting a cellwith an agent that functionally replaces one of the transcriptionfactor, it is possible to generate pluripotent cells with all of theabove-listed transcription factors except for the transcription factorreplaced or complemented by the agent.

III. Limiting the Development or Growth of Pluripotent Cells

Provided herein are methods of transdifferentiating an animal cell froma first non-pluripotent cell fate to a second non-pluripotent cell fate,e.g., by increasing the quantity of at least one reprogrammingtranscription factor in an animal cell having the first cell fate togenerate a cell that differentiates in response to lineage-specificdifferentiating factors. It was discovered that the methods providedherein do not require the generation of an intermediate, pluripotentcell. In fact, it has been now discovered that limiting the developmentor growth of pluripotent cells generally improves transdifferentiationefficiency, e.g., from a first non-pluripotent cell fate to a secondnon-pluripotent cell fate. Thus, in some embodiments, the methods of thepresent invention comprise limiting the development or growth ofpluripotent cells.

The development or growth of pluripotent cells can be limited in severalways. For example, the standard reprogramming protocol can be modified,e.g., by modifying reprogramming media. As described above,reprogramming media can be modified by omitting components that aregenerally necessary for significant pluripotent cell production, e.g.,LIF. In some embodiments, reprogramming media can be modified byomitting feeder MEFs. Alternatively, reprogramming media can be modifiedby including components that are harmful or detrimental for pluripotentcell production, e.g., FBS.

The development or growth of pluripotent cells can be limited by limitedexpression of transcription factors. As detailed in the examples herein,limited expression of transcription factors can be achieved by placingreprogramming transcription factors under the control of an induciblepromoter (e.g., a doxycycline-inducible promoter). For example,transcription factors can be silenced in the absence of doxycycline.Limited expression of transcription factors can be achieved by transienttransfection of reprogramming plasmids or other non-integratingreprogramming plasmids (see, e.g., Okita, K., et al. (2008) Science 322,949-953). Similarly, expression of exogenous transcription factors canbe eliminated, i.e., the cells are not transfected with a reprogrammingplasmid at all. For example, the transcription factors can be introducedby, e.g., culturing target cells in the presence of one or more proteinsunder conditions to allow for introduction of the proteins into thecell.

The development or growth of pluripotent cells can be limited by certaininhibitors that inhibit the growth of pluripotent cells. For example,certain inhibitors can be used to selectively inhibit the growth ofpluripotent cells, but not the growth of somatic cells. Exemplaryselective inhibitors include, but are not limited to, JAK inhibitors,PI3K inhibitors, CDK2 inhibitors, CDK4 inhibitors, and glycolysisinhibitors. These inhibitors can be used individually or in combination,to inhibit the growth of pluripotent cells.

JAK Inhibitors

Inhibitors of Janus Kinase (JAK) suitable for use in the presentinvention are well known in the art, see for example, U.S. Pat. No.6,452,005. In addition, bis monocyclic, bicyclic or heterocyclic arylcompounds (PCT Publication No. WO 92/20642), vinylene-azaindolederivatives (PCT Publication No. WO 94/14808) and1-cycloproppyl-4-pyridyl-quinolones (U.S. Pat. No. 5,330,992) have beendescribed the use of these agents as tyrosine kinase inhibitors. Styrylcompounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridylcompounds (U.S. Pat. No. 5,302,606), certain quinazoline derivatives(published EP Application No. 0 566 266 A1), seleoindoles and selenides(PCT Publication No. WO 94/03427), tricyclic polyhydroxylic compounds(PCT Publication No. WO 92/21660) have also been disclosed to betyrosine kinase inhibitors. An exemplary JAK inhibitor is2-(1,1-Dimethylethyl)-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinolin-7-one(“Ji1”).

PI3K Inhibitors

Phosphoinositide 3-kinases (PI 3-kinases or PI3Ks) are a family ofrelated intracellular single transducer enzymes capable ofphosphorylating the 3 position hydroxyl group of the inositol ring ofphosphatidylinositol (PtdIns or PI). These enzymes are also known asphosphatidylinositol-3-kinases. Based on based on primary structure,regulation, and in vitro lipid substrate specificity, thephosphoinositol-3-kinase family can be divided into three differentclasses: Class I, Class II and Class III (see Leevers et al., (1999)Current Op. Cell Biol. 11:219).

As used herein, the term “PI3K inhibitor” refers to a compound thatinhibits at least one activity of a PI3K of Class I, II or III on atleast one of its substrates (e.g., phosphorylating phosphatidylinositolto produce phosphatidylinositol 3-phosphate (PI(3)P),phosphatidylinositor (3,4)-bisphosphate (PI(3,4)P.sub.2), orphosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P.sub.3). A personskilled in the art can readily determine whether a compound, such aswortmannin or LY294002, is a PI3K inhibitor. A specific method ofidentifying such compounds or ligand is disclosed in, for example, U.S.Pat. Nos. 5,858,753; 5,882,910; and 5,985,589, Jackson et al. (2005)Nat. Med. 11:507, Pomel et al. (2006) J. Med. Chem. 49:3857; Palanki etal. (2007) J. Med. Chem. 50:4279), which methods are incorporated byreference herein.

In certain embodiments, a PI3K inhibitor inhibits the activity of aClass I PI3K. For example, a PI3K inhibitor may inhibit p110α, p110β, orp110γ, or p110Δ. In some embodiments, a PI3K inhibitor blocks or reducesthe activity of p110γ, or p110Δ as compared to untreated p110γ, orp110Δ. In certain embodiments, a PI3K inhibitor inhibits the activity ofa Class II PI3K. For example, a PI3K inhibitor may inhibit PI3K-C2α,PI3K-C2β, or PI3K-C2γ. In certain embodiments, a PI3K inhibitor inhibitsthe activity of a Class III PI3K, Vps34.

In certain embodiments, a PI3K inhibitor is selective or specific for aparticular PI3K isoform. An inhibitor is “selective” or “specific” for aparticular PI3K isoform if it inhibits that particular PI3K isoform moreeffectively than other PI3K isoforms. For example, an inhibitor specificfor a particular PI3K isoform may have an IC₅₀ for the particular PI3Kisoform at most about 1/10 (e.g., at most about 1/20, 1/30, 1/40, 1/50,1/60, 1/80, 1/100, 1/200, 1/300, 1/400, 1/500, 1/600, 1/800, or 1/1000)of the IC₅₀ for other PI3K isoforms. For example, a p110Δ-specificinhibitor may have an IC₅₀ value for p110Δ at most about 1/10 of theIC₅₀ for other PI3K isoforms (e.g., p110α, p110β, or p110γ).

In some embodiments, a PI3K inhibitor is specific for p110α, p110β, orp110γ, or p110Δ. In certain embodiments, a PI3K inhibitor inhibits twoor more classes or subclasses of PI3Ks. In certain embodiments, a PI3Kinhibitor is also an mTOR inhibitor.

Exemplary PI3K inhibitors include LY294002(2-morpholin-4-yl-8-phenylchromen-4-one) and wortmannin. Both LY294002and wortmannin are broad inhibitors against PI3K and can also inhibitmTOR. PI3K inhibitors also include wortmannin derivatives, such asPX-866 (see, Ihie et al., Mol Cancer Ther 3:763-72, 2004).

Exemplary p110γ-specific inhibitors include furan-2-ylmethylenethiazolidinediones (AS-252424) (see, Pomel et al., 2006, supra) and3,3′-(2,4-diaminopteridine-6,7-diyl)diphenol (see, Palanki et al.,supra). Exemplary p110Δ-specific inhibitors include IC486068 and IC87114(ICOS Corp., now Eli Lilly and Company) and CAL-101 and CAL-263(Calistoga Pharmaceuticals). Another PI3K inhibitor with p110Δ and p110βinhibition is CAL-120, a PI3K inhibitor (Calistoga Pharmaceuticals).Another exemplary PI3K inhibitor is GDC-0941 bismesylate(2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morphol-in-4-yl-thieno[3,2-d]pyrimidine,bimesylate salt), a p110α and P110Δ selective inhibitor.

Additional PI3K inhibitors include pyrazole derivatives disclosed in PCTPublication No. WO 2009/059030, amino triazole derivatives disclosed inWO 2009/068482, an imidazothiadiazole compound disclosed in WO2009/040552, fused pyrimidin-4-one compounds specific for p110Δdisclosed in WO 2009/064802, morpholino-pyrimidine compounds thatinhibit p110α disclosed in WO 2009/066084, a 4-pyrimidin-4-yl-morpholinederivative disclosed in WO 2009/042607, a4-morpholin-4-yl-thienopyrimidine compound disclosed in WO 2009/036082,a pyridosulphonamide derivative disclosed in WO 2009/055418,pyridopyrimidine derivatives that inhibit p110α and/or p110γ disclosedin WO 2009039140, heterocyclic derivatives that inhibit p110α disclosedin WO 2009046448, furanopyrimidines and zolopyrimidines specific forp110Δ disclosed in WO 2008/152394 and WO 2008/152390, quinazolinecompounds specific for p110Δ disclosed in WO 2008/152387,pyrimidine-substituted purine derivatives disclosed in WO 2009/045175,thienopyrimidine and pyrazolopyrimidine compounds disclosed in WO2009/052145, imidazolopyrimidine, pyrrolopyrimidine andpyrazolopyrimidine analogues disclosed in WO 2009/070524, substitutedimidazopyridazine disclosed in WO 2008/138834, thienopyrimidienederivatives selective for the p110Δ disclosed in WO 2009/053715, purinederivatives selective for the p110Δ disclosed in WO 2009/053716,2-(morpholin-4-yl)-substituted purine derivatives disclosed in WO2009/045174, PI3KΔ (p110Δ) inhibitors disclosed in U.S. Pat. Nos.6,518,277 and 6,800,620 and U.S. Application Publication No.2005/0261317, and BGT226, XL765 and BEZ235 (Novartis).

CDK2 Inhibitors

Illustrative cyclin-dependent kinase II (CDK2) inhibitors that may beemployed in the broad practice of the invention include thecyclin-dependent kinase II inhibitor compounds described in thefollowing references, the disclosures of all of which are herebyincorporated herein by reference in their respective entireties: (A)substituted oxindole derivatives described in International PatentApplication No. PCT/EP98/05559 filed Sep. 3, 1998 for “SubstitutedOxindole Derivatives,” (B) purine derivatives described in InternationalPublication WO97/20842; (C) pyridylpyrimidinamine derivatives describedin International Publication WO95/09852; (D) 2,6,9-trisubstitutedcompounds described in International Publication WO98/05335 of CVTherapeutics; (E) 4H-1-benzopyran-4-one derivatives described in GermanPatent 3836676; (F) 2-thiol and 2-oxo-flavopiridol analogues describedin U.S. Pat. No. 5,705,350, and in U.S. Pat. No. 5,849,733; (G)pyrido[2,3-D]pyrimidines and 4-aminopyrimidines described inInternational Publication WO98/33798 as well as in U.S. Pat. Nos.5,776,942; 5,733,913; 5,223,503; 4,628,089; 4,536,575; 4,431,805; and4,252,946; (H) antiviral CDK2 inhibitor compounds described inInternational Publication WO98/39007; (I) chimeric CDK2 inhibitorsdescribed in International Publication WO97/27297 of Mitotix Inc.; (J)the 2,6,9-trisubstituted purines described in Imbach, P., et al.,2,6,9-Trisubstituted Purines: Optimization Towards Highly Potent andSelective CDK1 Inhibitors, Bioorganic and Medicinal Chemistry Letters, 9(1999), 91-96; (K) the peptide inhibitors described in U.S. Pat. No.5,625,031 issued Apr. 29, 1997; (L) CDK2 inhibitor antisense sequencesdescribed in U.S. Pat. No. 5,821,234 issued Oct. 13, 1998; (M) the C2alkynylated purines described in Legraverend, M., et al., Synthesis ofC2 Alkynylated Purines, a New Family of Potent Inhibitors ofCyclin-Dependent Kinases, Bioorganic & Medicinal Chemistry Letters 8(1998) 793-798; and (N) the tyrphostins described in Kleinberger-Doron,N., et al., Inhibition of Cdk2 Activation by Selected Tyrphostins CausesCell Cycle Arrest at Late G1 and S Phase, Experimental Cell Research241, 340-351 (1998).

CDK4 Inhibitors

CDK4 (cyclin dependent kinase 4) inhibitors can, in the broadest sense,be any compound that is capable of inhibiting the activity of CDK4. Assuch, general CDK inhibitors that can inhibit the activity of two ormore different CDKs, e.g., CDK1, CDK2, as well as CDK4, can be used.Alternatively, CDK4 selective inhibitors can be used. It is nowunderstood that CDK4 and CDK6 are closely related kinases with virtuallyindistinguishable biochemical properties. The amino acid and nucleicacid sequence coding for human CDK4 and CDK6 can be found at Genbankaccession numbers NM 13 000075 and NM 13 001259, respectively. TheGenbank accession number U37022 also refers to CDK4. As used herein,CDK4 inhibitor refers to a compound that can be demonstrated to inhibitthe activity of CDK4 or of CDK6. A given inhibitor is considered to beselective for CDK4 if its determined inhibitory activity for CDK4 is atleast 5-fold, at least 10-fold, or at least 25-fold more potent than itsdetermined inhibitory activity for CDK that is other than CDK4 and CDK6,e.g., CDK1, CDK2, etc.

A number of assays are known in the art for determining CDK4 inhibitoryactivity of a compound, where representative such assays are describedin U.S. Pat. Nos., 6,040,321; 6,569,878; etc., where representative invitro assays that find use evaluate, in a time dependent manner, a givencompound's ability to inhibit the ability of CDK4 to incorporateradiolabeled phosphate donor into a protein substrate.

Representative specific CDK4 inhibitors include, but are not limited to,the following:

-   -   (i) naturally occurring indolocarbazole arcyriaflavin A as well        as substituted indolocarbazoles (see, e.g., Zhu et al., J Med.        Chem. 2003, 46, 2027-2030). Other derivatives of interest        include those described in U.S. Patent Application Publication        Nos. 2003/0229026 and 2004/0048915 (or equivalently, WO 01/44247        and WO 02/28861, respectively) which disclose        indolo[6,7-a]pyrrolo[3,4-c]carbazole-6,8-diones as potent CDK4        inhibitors.    -   (ii) semi-synthetic flavopiridol (also known as alvocidib)        disclosed in U.S. Pat. No. 4,900,727; as well as analogs of        flavopiridol, such as those reported in U.S. Pat. Nos. 5,733,920        and 5,849,733.    -   (iii) diarylurea derivatives disclosed in EP 1199306 A1, and        structurally related 2(1H)-pyrazinone fused aromatic or        heterocyclic derivatives having CDK4 and CDK6 activity described        in US 2003/0203907 (or equivalently, EP 1295878 A1).    -   (iv) diaminothiazoles, e.g., as reported in US 2002/0151554 (now        U.S. Pat. No. 6,756,374 B2), where a specific example of such        compounds is the compound denoted by the research code        Ro-0506220.    -   (v) napthyridinones disclosed in U.S. Pat. No. 6,150,359, the        pyrido[2,3-d]pyrimidines disclosed in U.S. Pat. No. 6,498,163        B1, and the 2-(pyridin-2-ylamino)pyrido[2,3-d]pyrimidin-7-ones        disclosed in patent publication US 2003/0149001. In particular,        US 2003/0149001 discloses        6-acetyl-8-cyclopentyl-5-methyl-2-[5-(1-piperazinyl)pyridin-2-ylamino]pyr-ido[2,3-d]pyrimidin-7(8H)-one        (7a) denoted by the research code PD-332991.    -   (vi) the pyrimidine derivatives disclosed in WO 00/12485 and        U.S. Pat. No. 6,593,326 B1, the imidazo[1,2-a]pyridine and        pyrazolo[2,3-a]pyridine derivatives disclosed in WO 01/14375,        the 4-amino-5-cyano-2-anilo-pyrimidine derivatives disclosed in        the publication US 2003/0087923 A1, the aminothiazole compounds        disclosed in U.S. Pat. Nos. 6,040,321, 6,262,096 B1, and        6,569,878 B1; the acridone and benzothiadiazine derivatives        disclosed in U.S. Pat. No. 6,630,464 B1 (or equivalently WO        98/49146); and the oxindole derivatives disclosed in U.S. Pat.        No. 6,720,332 B2 (or equivalently WO 02/20524). Yet further        examples of CDK4 inhibitors are described in Toogood, Curr.        Opin. Cell. Biol. 2002, 6, 472-478, Toogood, Med. Res. Rev.,        2001, 21, 487-498, and in Carini et al., Bioorgan. Med. Chem.        Lett., 2001, 11, 2209-2211.

Glycolysis Inhibitors

Enzymes associated with glycolysis pathway are known in the art andinclude hexokinase, glucokinase (in tumors or in rapidly proliferatingtissues), phosphoglucose isomerase, phosphofructokinase, aldolase,triose phosphate isomerase, glyceraldehydes 3-phosphate dehydrogenase,phosphoglycerate kinase, phosphoglyceromutase, enolase, pyruvate kinaseand, indirectly, lactate dehydrogenase (lactate metabolism). It is anaspect of the invention that an inhibitor of anaerobic ATP synthesis isan inhibitor of an enzyme associated with the glycolytic pathway.Inhibitors of the glycolytic pathway are any known in the art.

Inhibitors of hexokinase may be configurational isomers ofmonosaccharides modified at C-6 by substitution (replacement) or removalof 6-OH (the hydroxyl group). An example of a substituent is a blockingmoiety—for example, an atom from the halogen family, such as fluorine(6-fluoro-D-glucose). Another example of a substituent is a thiol group.Monosaccharides modified at C-6 by removal of 6-OH will not betransformed by hexokinase or glucokinase (see below) toglucose-6-phosphate and can potentially block both enzymes.

Inhibitors of hexokinase are any known in the art and may include, butare not limited to any of the following:

-   -   (i) 6-fluoro-D-glucose, 6-bromo-D-glucose, 6-chloro-D-glucose,        6-O-methyl-D-glucose, 6-Thio-D-glucose, 6-deoxy-D-glucose, and        any derivative known in the art.    -   (ii) C-6 substituted derivatives of other hexose ring pyranoses        (mannopyranoses, galactopyranoses). Examples include        6-deoxy-6-fluoro-D-mannose, and any known in the art.    -   (iii) Various halogenated (fluoro, bromo, chloro-) C6 sugars        derivatives such as gluconolactones, glucuronic acid,        glucopyranoside, and their phosphate derivatives, and any known        in the art. Halogenated glucosides may also be delivered        indirectly to the cell, by compounds such as glucoronides with        halogenated glycosides at the C-1 position (once in the cell,        glucoronidases will cleave it, and deliver active hexose in the        cell). Preferably, an inhibitor of hexokinase is        6-deoxy-6-fluoro-D-glucose and its derivatives.

Inhibitors of glucokinase may be any in the art. They include, but arenot limited to mannoheptulose, mannoheptose, glucoheptose,N-acetylglucosamine. Glucokinase is predominantly present in tumoursonly.

Inhibitors of phosphoglucoisomerase: Phosphoglucoseisomerase transformsglucose 6-phosphate to fructose 6-phosphate. Such transformationrequires the presence of an hydroxyl group at C-2. Therefore, analogswithout hydroxyl or having the hydroxyl properly blocked will notundergo isomerization by phosphoglucose isomerase.

Another way to inhibit isomerization by phosphoglucose isomerase is bymodifying the glucose 6-phosphate at C-1 or C-5 by substituting hydroxylwith a halogenated atom (fluorine, glucosyl fluoride), or by simpledeoxygenation to 1-deoxy-D-glucose.

Inhibitors of phosphoglucoisomerase are any known in the art and mayinclude, but are not limited to any of the following:

-   -   (i) C2 substituted D-hexoses, such as        2-deoxy-2-halogeno-D-hexoses, such as 2-deoxy-2-fluoro-D-glucose        (2FDG), 2-chloro-2-deoxy-D-glucose, 2-bromo-D-glucose,        2-iodo-D-glucose, 2-deoxy-2,2-difluoro-D-arabino-hexose,        2-deoxy-2-fluoro-D-mannose, 2-deoxy-D-arabino-hexose,        2-Deoxy-2-fluoro-D-galactose,        1,6-anhydro-2-deoxy-2-fluoro-beta-D-glucopyranose        (1-6-anhydrosugar), 2-amino-2-deoxy-D-glucose (glucose amine),        2-amino-2-deoxy D galactose (galactosamine),        2-amino-2-deoxy-D-mannose (mannosamine),        2-deoxy-2-fluoro-D-mannose, 2-deoxy-2-fluoro-D-galactose,        2-deoxy-D-arabino-hexose, 2-deoxy-2,2-difluoro-D-arabino-hexose,        2-deoxy-2-fluoro-D-glucose 1-Phosphate,        2-deoxy-2-fluoro-D-glucose 6-P, 2-deoxy-2-fluoro-D-glucose 1,6        biphosphate, 2-deoxy-2-fluoro-D-mannose 1-P,        2-deoxy-2-fluoro-D-mannose 6-P, 2-deoxy-2-fluoro-D-mannose        1,6-biphosphate, nucleotide diphosphate (for example uridine        di-P)-2deoxy-2-fluoro-D-glucose, mannose.    -   (ii) C-2-halogen substituted, and NH3 substituted derivatives of        D-Glucose 6-phosphate, 2-deoxy-2-fluoro-2-D-glucose-6-phosphate,        2-chloro-2-deoxy-D-glucose-6-phosphate,        2-deoxy-D-arabino-hexose-6-phosphate, D-glucosamine-6-phosphate,        2-deoxy-2-fluoro-2-D-manose-6-P, and any known derivatives.    -   (iii) C-2 halogenated derivatives of hexose ring pyranoses        (mannopyranoses, galactopyranoses), for instance        C-2-deoxy-2-fluoro-D-pyranoses, and any known in the art.    -   (iv) Halogenated (fluoro, bromo, chloro, iodo) C2 sugars        derivatives such as gluconolactones, glucuronic acid,        glucopyranoside, and their phosphate derivatives.    -   (v) Modification at C-1 or C-5: replacement of hydroxyl by        fluorine or deoxygenation or replacement by a sulfur group in        C-5, such as but not limited to glucosyl fluoride,        1-deoxy-D-glucose, 5-thio-D-glucose.    -   (vi) 6-aminonicotinamide (6AN), indirectly by the inhibition of        the PPP.

Inhibitors of phosphofructokinase (or fructose-6-P kinase) are any knownin the art and may include, but are not limited to any of the following:Acidosis-inducing agents, 2-deoxy-2-fluoro-D-glucose, citrate andhalogenated derivatives of citrate, fructose 2,6-bisphosphate,bromoacetylethanolamine phosphate analogues(N-(2-methoxyethyl)-bromoacetamide, N-(2-ethoxyethyl)-bromoacetamide,N-(3-methoxypropyl)-bromoacetamide).

Inhibitors of aldolase: Analogs blocking the aldolase cleavage, thusblocking formation of trioses from fructose 1,6-bisphosphate require thepresence of hydroxyl groups at C-3 and C-4. Thus, for example, 3-deoxyor 3-fluoro-D-glucose or 4-deoxy or 4-fluoro-D-glucose can betransformed to 4-fluoro-D-fructose 1,6-bisphosphate, which will not becleaved by aldolase but will block it.

Inhibitors of glyceraldehyde 3P deshydrogenase are any known in the artand may include, but are not limited to any of the following:Iodoacetate, pentalenolactone, arsenic,1,1-difluoro-3-phosphate-glycerol.

Inhibitors of the transformation chain of glyceraldehyde (glyceraldehyde3P deshydrogenase, phosphoglycerate kinase, phosphoglycerate mutase,enolase) are any which act at any step where phosphorylation isinvolved. Such inhibitors are any known in the art and may include, butare not limited to any of the following: either 2-fluoro (or iodo, orthio, or methoxy) or 3-fluoro (or 3,3 difluoro, 3-iodo, 3-carboxylo-,3-thio)-glyceraldehydes or glycerates, 3-fluoro-2-phosphoglycerate,also, phosphothioesters or other phosphorous-modified analogs can blockthe transformations of glyceraldehyde.

Inhibitors of pyruvate kinase are any known in the art. Alternatively, acomposition comprising serine or fructose 1,6-diP shifts the glycolyticpathway towards the TCA cycle; thus a composition of the inventioncomprises serine and an inhibitor of the TCA such as fluoroacetate or aninhibitor of the oxidative phosphorilation such as rhodamine.

Inhibitors of pyruvate carboxylase and PEP carboxylase, triose phosphateisomerase, phosphoglycerate kinase, enolase, phosphoglycerate mutase andtriose phosphate isomerase are any known in the art.

Inhibitors of lactate deshydrogenase are any known in the art and mayinclude, but are not limited to oxamate, 2-fluoro-propionic acid or itssalts; 2,2-difluoro-propionic acid, pyruvate modified at C-3 such as,but not limited to 3-halo-pyruvate, 3-halopropionic acid and2-thiomethylacetic acid.

In some embodiments, an inhibitor of glycolysis is any of 2FDG, oxamateand iodoacetate.

Glycolysis is the main pathway for anaerobic ATP synthesis. Tumoursswitch to anaerobic ATP synthesis by metabolizing the well-distributedglucose among others in order to provide nucleotides through the PPPpathway. It is known that proliferating masses which are partly underanaerobic type respiration are more resistant to radiation orchemotherapy. Therefore, by locally inhibiting the glycolysis pathway,anaerobic respiration which is the principal energy pathway of poorlyoxygenated cells is inhibited, leading to increased cell death ofhypoxic proliferating cells. The proliferation of non-hypoxic cells isslowed as well owing to the shutdown of this primary energy pathway.

IV. Generation of Cells Having a Second Cell Fate

In one aspect, the present invention provides methods fordifferentiating cells into a desired cell fate. As shown in theExamples, the inventors can generate differentiated cells, i.e., cellshaving a second cell fate, of any of the three major lineages (endoderm,mesoderm, ectoderm). A number of differentiation protocols (i.e.,protocols comprising contacting cells with lineage-specificdifferentiation factors) are known for differentiating cells into cellfates and it is believed that such protocols can generally be applied tocells generated as described above in sections II and III to generatedifferentiated cells. In some embodiments, the differentiation protocolincludes only chemically defined media.

In some embodiments, the methods of sections II and III are performedcontinuously, i.e., without significant intervening steps. For example,in some embodiments, transition between steps simply involves a changeof media.

A. Mesodermal Differentiation

In some embodiments, cells are differentiated into mesodermal cells. Insome embodiments, cells are differentiated into cardiac cells (e.g.,cardiomyocytes). In combination with the methods described above insections II and III, it is believed that one can use any method forinducing cardiomycytes as desired. In some embodiments, the conditionsinclude a chemically defined medium. In some embodiments, non-mesodermalcells (e.g., ectodermal cells, e.g., fibroblasts) are exposed (e.g., viaexpression or protein contact) to reprogramming transcription factorsfor, e.g., 3-10, 3-6 days, etc., optionally in the presence of a JAKinhibitor (e.g., at a sufficient concentration to prevent development ofiPS cells). At the end of this period, cells can be (e.g., directly)moved to conditioning media to convert the cells to mesodermal cells asdescribed herein.

In some embodiments, the cardiomyocytes are induced by contacting a cell(for example, a cell as generated in section II and/or III above, or ifdesired, an induced pluripotent cell or other pluripotent or progenitorcell) with a BMP protein, a calcium channel agonist, a Gαs activatingagent, a cAMP analog, and/or a GSK-3 inhibitor under conditions togenerate cardiomyocytes. In some embodiments, the cardiomyocytes areinduced by contacting a cell (for example, a cell as generated insection II and/or III above, or if desired, an induced pluripotent cell,other pluripotent or progenitor cell) with bone morphogenic protein-4(BMP4) and/or an GSK-3 inhibitor under conditions to generatecardiomyocytes. In some embodiments, the conditions include a chemicallydefined medium. In some embodiments, for example, a chemically definedmedium includes RPMI-1640 supplemented with 0.5×N2, 1× B27 (withoutvitamin A), 0.05% BSA fraction V, 0.5% Glutamax, and 0.1 mMβ-mercaptoethanol (Invitrogen).

The BMP protein (e.g., BMP4) applied to the cells can be from any of anumber of organisms, though in many embodiments, the species origin ofthe BMP protein (e.g., BMP4) will match the species of cell used. Thusfor example, in some embodiments, a human cell is contacted with humanBMP protein (e.g., BMP4), a mouse cell is contacted with mouse BMPprotein (e.g., BMP4), etc. It will be appreciated that active mutantsand fragments of BMP protein (e.g., BMP4) can also be used.

In some embodiments, the cardiomyocytes can be induced by contacting acell (for example, a cell as generated in section II and/or III above,or if desired an induced pluripotent cell or other pluripotent orprogenitor cell) with bone morphogenic protein-4 (BMP4) without an GSK-3inhibitor under conditions to generate cardiomyocytes. In someembodiments, the cardiomyocytes can also be induced by contacting a cellwith a GSK-3 inhibitor without BMP4.

In some embodiments, the cardiomyocytes are induced by contacting a cell(for example, a cell as generated in section II and/or III above, or ifdesired an induced pluripotent cell or other pluripotent or progenitorcell) with a L-type calcium channel agonist. Exemplary L-type calciumchannel agonists include, but are not limited to, BayK8644 (see, e.g.,Schramm, et al., Nature 303:535-537 (1983)), Dehydrodidemnin B (see,e.g., U.S. Pat. No. 6,030,943), FPL 64176 (FPL) (see, e.g., Liwang, etal., Neuropharmacology 45:281-292 (2003)), S(+)-PN 202-791 (see, e.g.,Kennedy, et al., Neuroscience 49:937-44 (1992)) and CGP 48506 (see,e.g., Chahine, et al., Canadian Journal of Physiology and Pharmacology81:135-141 (2003)).

In some embodiments, the cardiomyocytes are induced by contacting a cell(for example, a cell as generated in section II and/or III above, or ifdesired an induced pluripotent cell or other pluripotent or progenitorcell) with a Gαs activating agent. Exemplary Gαs activating agentsinclude, but are not limited to, isoproterenol, epinephrine, cimaterol,clenbuterol, dobutamine, alprenolol, cyanopindolol, propanolol, sotalol,timolol, and ICI-118,551(3-isoprophylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (see, e.g.,U{hacek over (g)}ur et al., Mol Pharmacol 68:720-28 (2005)).

In some embodiments, the cardiomyocytes are induced by contacting a cell(for example, a cell as generated in section II and/or III above, or ifdesired an induced pluripotent cell or other pluripotent or progenitorcell) with a cAMP analog. Exemplary cAMP analogs include, but are notlimited to, dibutyryl cAMP, 8-Bromo-cAMP, and Sp-8-Br-cAMPS(8-bromoadenosine-3′,5′-cyclic monophosphorothioate, Sp-isomer) (see,e.g., Ferrier and Howlett, J. Pharmacol. Exp. Ther. 306:166-76 (2003)).

The GSK-3 inhibitor used in the methods of mesodermal differentiationdescribed herein can be a GSK-3 isoform specific inhibitor, e.g., aGSK-3β or a GSK-3α inhibitor, or a isoform non-specific inhibitor. Insome embodiments, the GSK-3 inhibitor can be substituted with a GSKinhibitor. In some embodiments, the GSK-3 inhibitor can be substitutedwith a Wnt inhibitor or a Wnt pathway inhibitor.

Wnt Inhibitors

By “Wnt pathway” is meant to include any of the proteins downstream orupstream of Wnt protein activity. For example, this could include LRP5,LRP6, Dkk, GSK-3, Wnt10B, Wnt6, Wnt3 (e.g., Wnt 3A), Wnt1 or any of theother proteins discussed herein, and the genes that encode theseproteins. Discussion of the Wnt pathway also is meant to include all ofthe pathways downstream of Wnt, such as the LRP5 or HBM pathways, theDkk pathway, the β-catenin pathway, the MAPKAPK2 pathway, the OPG/RANKpathway, and the like. By “LRP5 pathway” and “IBM pathway” is meant anyproteins/genes including LRP5 or the HBM mutant and proteins downstreamof LRP5 or the HBM mutant. By “β-catenin pathway” is meant anyproteins/genes including β-catenin and proteins downstream of β-catenin.By “MAPKAPK2 pathway” is meant any proteins/genes including MAPKAPK2 andproteins downstream of MAPKAPK2. By “OPG/RANKL pathway” is meant anyproteins/genes including OPG/RANKL and proteins downstream of OPG andRANKL. By “Dkk pathway” is meant to include any proteins/genes involvedin Dkk-1 and LRP5 and/or LRP6 interaction that is part of the Wntpathway. Dkk-1 inhibits LRP5 activity.

Exemplary Wnt inhibitors include, but are not limited to, e.g., IWP-2(2-(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-ylthio)-N-(6-methylbenzo[d]thiazol-2-yl)acetamide),DKK1 (Dickkopf protein 1), and IWR1

IWP-2 was identified in a high throughput screen for antagonists of theWnt/β-catenin pathway (Chen, et al., Nat Chem Biol 5: 100-7, 2009). WntInhibitor IWP-2 prevents palmitylation of Wnt proteins by Porcupine(Porcn), a membrane-bound O-acyltransferase, thereby blocking Wntsecretion and activity. It has an IC₅₀ of 27 nM and blocksphosphorylation of the Lrp6 receptor and accumulation of both Dvl2 andβ-catenin (Chen, et al., Nat Chem Biol 5: 100-7, 2009). Wnt inhibitorIWR1 induces an increase in Axin2 protein levels; promotes β-cateninphosphorylation by stabilizing Axin-scaffolded destruction complexes(Chen, et al., Nat Chem Biol 5: 100-7, 2009; Lu et al., Bioorg. Med.Chem. Lett. 19:3825, 2009). Additional Wnt inhibitors include, but arenot limited to, IWR compounds, IWP compounds and other Wnt inhibitorsdescribed in WO09155001 and Chen, et al., Nat Chem Biol 5: 100-7, 2009.

Known antagonists of Wnt signaling also include Dickkopf proteins,secreted Frizzled-related proteins (sFRP), Wnt Inhibitory Factor 1(WIF-1), and Soggy. Members of the Dickkopf-related protein family(Dkk-1 to −4) are secreted proteins with two cysteine-rich domains,separated by a linker region. Dkk-3 and -4 also have one prokineticindomain. Dkk-1, -2, and -4 function as antagonists of canonical Wntsignaling by binding to LRP5/6, preventing LRP5/6 interaction withWnt-Frizzled complexes. Dkk-1, -2, and -4 also bind cell surfaceKremen-1 or -2 and promote the internalization of LRP5/6. Antagonisticactivity of Dkk-3 has not been demonstrated. Dick proteins have distinctpatterns of expression in adult and embryonic tissues and have a widerange of effects on tissue development and morphogenesis. The Dickfamily also includes Soggy, which is homologous to Dkk-3 but not to theother family members. The sFRPs are a family of five Wnt-bindingglycoproteins that resemble the membrane-bound Frizzleds. The largestfamily of Wnt inhibitors, they contain two groups, the first consistingof sFRP-1, 2, and 5, and the second including sFRP-3 and 4. All aresecreted and derived from unique genes, none are alternate splice formsof the Frizzled family. Each sFRP contains an N-terminal cysteine-richdomain (CRD). Other antagonists of Wnt signaling include WIF-1 (WntInhibitory Factor 1), a secreted protein that binds to Wnt proteins andinhibits their activity.

GSK Inhibitor

By “GSK inhibitor” is meant any agent which inhibits GSK activity. Thesecan include non-selective GSK inhibitors, such as LiCl or other lithiumsalts, as well as selective GSK inhibitors. In some embodiments, GSKinhibitors are GSK-3 inhibitors. In some embodiments, GSK inhibitors areGSK-3 isoform specific inhibitors, such as GSK-3β or GSK-3α inhibitors.Additional inhibitors include but are not limited to monoclonal orpolyclonal antibodies or immunogenically active fragments thereof,peptide aptamers, a GSK binding protein, an antisense molecule to a GSKnucleic acid, an RNA interference molecule, a morpholinooligonucleotide, a peptide nucleic acid (PNA), a ribozyme, and apeptide.

As noted in the example, in some embodiments, the GSK-3 inhibitor is BIO(6-bromoindirubin-3′-oxime) as follows:

In some embodiments, the GSK-3 inhibitor is a BIO derivative havinggeneral formula:

In the Formula (I), —X and —Y is —H or a halogen, e.g., —Br. R₁ isselected from the group consisting of oxo,

R₂, R₃, R₄ are independently hydrogen, substituted or unsubstitutedalkyl (e.g., substituted or unsubstituted C₁ to C₂₀ alkyl), substitutedor unsubstituted heteroalkyl (e.g., substituted or unsubstituted 2 to 20membered heteroalkyl), substituted or unsubstituted cycloalkyl (e.g., C₃to C₁₄ cycloalkyl including fused ring structures), substituted orunsubstituted heterocycloalkyl (e.g., 3 to 14 membered heterocycloalkylincluding fused ring structures), substituted or unsubstituted aryl(e.g., a C₆ to C₁₄ aryl including fused ring structures), or substitutedor unsubstituted heteroaryl (e.g., 5 to 14 membered heteroaryl includingfused rings structures). Additional BIO derivatives include, but are notlimited to, those described in U.S. Publication No. 2007/0276025. Insome embodiments, the GSK-3 inhibitor is an indirubin moleculesubstituted with a halogen at position C6 of the indirubin molecule. Insome embodiments, the GSK-3 inhibitor is selected from the groupconsisting of 6-bromoindirubin, 6,6′-dibromoindimibin,6-bromoindirubin-3′-oxime (“BIO”), 6,6′-dibromnoindirubin-3′-oxime,6-bromoindirubin-3′-methoxime, 6-bromo-5-methylindirubin and6-bromoindirubin-3′-acetoxime, 6-bromo-5-aminoindirubin and6-bromo-5-amino-3′-oxime-indirubin, 6-bromoindirubin,6,6′-dibromoindirubin, 6-bromoindirubin-3′-oxime (“BIO”),6,6′-dibromoindirubin-3′-oxime, 6-bromoindirubin-3′-methoxime,6-bromo-5-methylindirubin, 6-bromo-5-aminoindirubin,6-bromo-5-amino-3′-oxime-indirubin, 6-bromoindirubin-3′-acetoxime,5-amino-indirubin, 5-amino-3′-oxime-indirubin, and pharmaceuticallyacceptable salts thereof.

Other possible inhibitors of GSK-3 can include antibodies that bind,dominant negative variants of, and siRNA, microRNA, antisense nucleicacids, and other polynucleotides that target GSK-3. Specific examples ofGSK-3 inhibitors include, but are not limited to, Kenpaullone,1-Azakenpaullone, CHIR99021, CHIR98014, AR-A014418 (see, e.g., Gould, etal., The International Journal of Neuropsychopharmacology 7:387-390(2004)), CT 99021 (see, e.g., Wagman, Current Pharmaceutical Design10:1105-1137 (2004)), CT 20026 (see, Wagman, supra), SB216763 (see,e.g., Martin, et al., Nature Immunology 6:777-784 (2005)), AR-A014418(see, e.g., Noble, et al., PNAS 102:6990-6995 (2005)), lithium (see,e.g., Gould, et al., Pharmacological Research 48: 49-53 (2003)), SB415286 (see, e.g., Frame, et al., Biochemical Journal 359:1-16 (2001))and TDZD-8 (see, e.g., Chin, et al., Molecular Brain Research,137(1-2):193-201 (2005)). Further exemplary GSK-3 inhibitors availablefrom Calbiochem (see, e.g., Dalton, et al., WO2008/094597, hereinincorporated by reference), include but are not limited to BIO(2′Z,3′£)-6-Bromoindirubin-3′-oxime (GSK-3 Inhibitor IX); BIO-Acetoxime(2′Z,3′E)-6-Bromoindirubin-3′-acetoxime (GSK-3 Inhibitor X);(5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine(GSK-3-Inhibitor XIII); Pyridocarbazole-cyclopenadienylruthenium complex(GSK-3 Inhibitor XV); TDZD-84-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (GSK-3β Inhibitor I);2-Thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK-3β InhibitorII); OTDZT 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione (GSK-3β InhibitorIII); alpha-4-Dibromoacetophenone (GSK-3β Inhibitor VII); AR-AO 14418N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (GSK-3β InhibitorVIII);3-(1-(3-Hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione(GSK-3β Inhibitor XI); TWS1 19 pyrrolopyrimidine compound (GSK-3βInhibitor XII); L803 H-KEAPPAPPQSpP-NH2 or its Myristoylated form(GSK-3β Inhibitor XIII); 2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone(GSK-3β Inhibitor VI); AR-AO144-18; SB216763; and SB415286. Residues ofGSK-3β that interact with inhibitors have been identified. See, e.g.,Bertrand et al., J. Mol Biol. 333(2): 393-407 (2003).

Those of skill will appreciate that the concentration of the GSK-3inhibitor will depend on which specific inhibitor is used. In certainembodiments, a combination of two or more different GSK-3 inhibitors canbe used.

B. Ectodermal Differentiation

In some embodiments, cells are differentiated into ectodermal cells. Insome embodiments, cells are differentiated into neural cells. In someembodiments, cells are differentiated into neural progenitor cells. Insome embodiments, cells are differentiated into tyrosine hydroxylase(TH)-positive neuronal cells. In some embodiments, cells aredifferentiated into retinal pigmented epithelium cells. In combinationwith the methods described above in sections II and III, it is believedthat one can use any method for inducing differentiation into ectodermalcells as desired. In some embodiments, the conditions include achemically defined medium. In some embodiments, non-ectodermal cells areexposed (e.g., via expression or protein contact) to reprogrammingtranscription factors for, e.g., 3-10, 3-6 days, etc., optionally in thepresence of a JAK inhibitor (e.g., at a sufficient concentration toprevent development of iPS cells). At the end of this period, cells canbe (e.g., directly) moved to conditioning media to convert the cells toectodermal cells as described herein.

In some embodiments, differentiation into neural cells is induced bycontacting a cell (for example, a cell as generated in section II and/orIII above, or if desired an induced pluripotent cell or otherpluripotent or progenitor cell) with a Wnt inhibitor or a Wnt pathwayinhibitor as used in the methods of mesodermal differentiation (e.g.,IWP-2, DKK1, and IWR1) under conditions to generate neural cells. Insome embodiments, neural cells (e.g., neural progenitor cells) areinduced by contacting a cell (for example, a cell as generated insection II and/or III above, or if desired an induced pluripotent cellor other pluripotent or progenitor cell) with FGF2, FGF4, and EGF underconditions to generate neural cells (e.g., neural progenitor cells). Insome embodiments, neural cells (e.g., TH-positive neuronal cells) areinduced by contacting a cell (for example, a cell as generated insection II and/or III above, or if desired an induced pluripotent cellor other pluripotent or progenitor cell) with FGF8 and SHH underconditions to generate neural cells (e.g., TH-positive neuronal cells).In some embodiments, the conditions include a chemically defined medium.In some embodiments, a chemically defined medium includes an AdvancedDMEM/F12 and Neurobasal 1:1 mixture supplemented with 0.05% bovine serumalbumin, 1×N2, 1× B27, 2 mM Glutamax, and 0.11 mM β-mercaptoethanol.

In some embodiments, differentiation into retinal pigmented epitheliumcells is induced by contacting a cell (for example, a cell as generatedin section II and/or III above, or if desired an induced pluripotentcell or other pluripotent or progenitor cell) with FGF2, FGF4, and EGFunder conditions to generate a neural progenitor cell (e.g., asdescribed above), and contacting the neural progenitor cell with a TGFβinhibitor and a GSK-3 inhibitor under conditions to generate the retinalpigmented epithelium cell. In some embodiments, the conditions include achemically defined medium.

In some embodiments, growth factors generally known and used in celldifferentiation, e.g., neural cell differentiation, can be used forectodermal differentiation according to the present invention. In someembodiments, the method involves contacting cells with FGF2, FGF4 and/orEGF to generate neural cells. In some embodiments, the method involvescontacting cells with FGF8 and SHH to generate neural cells.

In some embodiments, GSK-3 inhibitors, for example a GSK-3 isoformspecific inhibitor, e.g., a GSK-3β or a GSK-3α inhibitor, or a isoformnon-specific inhibitor, can be used for ectodermal differentiation(e.g., retinal pigmented epithelium cell differentiation) according tothe present invention. In some embodiments, the GSK-3 inhibitor can besubstituted with a GSK inhibitor. In some embodiments, the GSK-3inhibitor can be substituted with a Wnt inhibitor or a Wnt pathwayinhibitor. Suitable GSK-3 inhibitors and/or Wnt inhibitors and/or Wntpathway inhibitors are described above. In some embodiments, a TGFβinhibitor can be used for ectodermal differentiation (e.g., retinalpigmented epithelium cell differentiation) according to the presentinvention. Suitable TGFβ inhibitors are described infra.

C. Endodermal Differentiation

In some embodiments, cells are differentiated into endodermal cells. Insome embodiments, cells are differentiated into pancreatic cells (e.g.,pancreatic lineage cells or pancreatic beta cells). In some embodiments,cells are differentiated into induced definitive endoderm (iDE) cells.In combination with the methods described above in sections II and III,it is believed that one can use any method for inducing differentiationinto endodermal cells as desired. In some embodiments, the conditionsinclude a chemically defined medium. In some embodiments, non-endodermalcells (e.g., ectodermal cells, e.g., fibroblasts) are exposed (e.g., viaexpression or protein contact) to reprogramming transcription factorsfor, e.g., 3-10, 3-6 days, etc., optionally in the presence of a JAKinhibitor (e.g., at a sufficient concentration to prevent development ofiPS cells). At the end of this period, cells can be (e.g., directly)moved to conditioning media to convert the cells to endodermal cells asdescribed herein.

In some embodiments, differentiation into pancreatic cells is induced bycontacting a cell (for example, a cell as generated in section II and/orIII above, or if desired an induced pluripotent cell or otherpluripotent or progenitor cell) with a TGFβ/Activin/Nodal pathwayinhibitor (e.g., A-83-01 or SB431542) under conditions to generatepancreatic cells. In some embodiments, differentiation into pancreaticlineage cells is induced by contacting a cell (for example, a cell asgenerated in section II and/or III above, or if desired an inducedpluripotent cell or other pluripotent or progenitor cell) with aTGFβ/Activin/Nodal family member optionally in the presence of a JAKinhibitor, then contacting the cell with a TGFβ/ALK5 inhibitor (e.g.,SB431542), then contacting the cell with nicotinamide under conditionsto generate the pancreatic lineage cell.

In some embodiments, differentiation into an induced definitive endoderm(iDE) cell is induced by contacting a cell (for example, a cell asgenerated in section II and/or III above, or if desired an inducedpluripotent cell or other pluripotent or progenitor cell) with aTGFβ/Activin/Nodal pathway inhibitor (e.g., A-83-01 or SB431542), anHDAC inhibitor (e.g., NaB), and/or a GSK-3 inhibitor (e.g., CHIR99021)under conditions to generate the iDE cell. In some embodiments,differentiation into a pancreatic beta cell is induced by contacting acell (for example, a cell as generated in section II and/or III above,or if desired an induced pluripotent cell or other pluripotent orprogenitor cell) with a TGFβ/Activin/Nodal pathway inhibitor (e.g.,A-83-01 or SB431542), an HDAC inhibitor (e.g., NaB), and/or a GSK-3inhibitor (e.g., CHIR99021) under conditions to generate the iDE cell,then under conditions to generate pancreatic beta cells, contacting thecell with FGF7, retinoic acid (RA), a Hedgehog pathway inhibitor (e.g.,GDN-0449), a BMP inhibitor (e.g., LDN-193189), and/or a TGFβ/ALK5inhibitor (e.g., SB431542), then contacting the cell with EGF and/or aNotch inhibitor (e.g., DAPT), then contacting the cell with bFGF,nicotinamide, and/or extendin-4.

In some embodiments, the conditions include a chemically defined medium.In some embodiments, for example, a chemically defined medium includesRPMI-1640 supplemented with 0.5×N2, 1× B27 (without vitamin A), 0.05%BSA fraction V, 0.5% Glutamax, and 0.1 mM β-mercaptoethanol(Invitrogen).

Growth factors generally known and used in cell differentiation, e.g.,pancreatic cell differentiation, can be used for endodermaldifferentiation accordingly to the present invention. In someembodiments, the method involves contacting cells with bFGF, BMP4, EGF,and/or FGF7 to generate endodermal cells.

Small molecules generally known and used in cell differentiation, e.g.,pancreatic cell differentiation, can be used for endodermaldifferentiation accordingly to the present invention. In someembodiments, pancreatic cell differentiation is induced by incubatingthe cells (for example, a cell as generated in section II and/or IIIabove, or if desired an induced pluripotent cell, other pluripotent orprogenitor cell) with a sufficient amount of a GSK inhibitor, an HDACinhibitor and/or a TGFβ/activin pathway inhibitor (e.g., A-83-01).

TGFβ/Activin/Nodal Pathway Inhibitors

Exemplary TGFβ/activin pathway inhibitors include but are not limitedto: TGF beta receptor inhibitors, inhibitors of SMAD 2/3phosphorylation, inhibitors of the interaction of SMAD 2/3 and SMAD 4,and activators/agonists of SMAD 6 and SMAD 7. Furthermore, thecategorizations described below are merely for organizational purposesand one of skill in the art would know that compounds can affect one ormore points within a pathway, and thus compounds may function in morethan one of the defined categories.

Inhibitors of SMAD 2/3 phosphorylation can include antibodies to,dominant negative variants of and antisense nucleic acids that targetSMAD2 or SMAD3. Specific examples of inhibitors include PD169316;SB203580; SB-431542; LY364947; A77-01; and3,5,7,2′,4′-pentahydroxyflavone (Morin). (See, e.g., Wrzesinski, supra;Kaminska, supra; Shimanuki, et al., Oncogene 26:3311-3320 (2007); andKataoka, et al., EP1992360, incorporated herein by reference.)

Inhibitors of the interaction of SMAD 2/3 and SMAD 4 can includeantibodies to, dominant negative variants of and antisense nucleic acidsthat target SMAD2, SMAD3 and/or smad4. Specific examples of inhibitorsof the interaction of SMAD 2/3 and SMAD4 include but are not limited toTrx-SARA, Trx-xFoxH1b and Trx-Lef1. (See, e.g., Cui, et al., Oncogene24:3864-3874 (2005) and Zhao, et al., Molecular Biology of the Cell,17:3819-3831 (2006).)

Activators/agonists of SMAD 6 and SMAD 7 include but are not limited tonucleic acids encoding SMAD 6 or SMAD 7 proteins or fragments thereofand polypeptides and fragments encoded by said nucleic acids. (See,e.g., Miyazono, et al., U.S. Pat. No. 6,534,476, incorporated herein byreference.)

In other embodiments, the differentiation into pancreatic cells areinduced by contacting a cell (for example, a cell as generated insection II and/or III above, or if desired an induced pluripotent cell,other pluripotent or progenitor cell) with a TGFβ receptor/ALK5inhibitor under conditions to generate pancreatic cells. TGF betareceptor (e.g., ALK5) inhibitors can include antibodies to, dominantnegative variants of, and antisense nucleic acids that suppressexpression of, TGF beta receptors (e.g., ALK5). Exemplary TGFβreceptor/ALK5 inhibitors include, but are not limited to, SB431542 (see,e.g., Inman, et al., Molecular Pharmacology 62(1):65-74 (2002)),A-83-01, also known as3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide(see, e.g., Tojo, et al., Cancer Science 96(11):791-800 (2005), andcommercially available from, e.g., Toicris Bioscience);2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine,Wnt3a/BIO (see, e.g., Dalton, et al., WO2008/094597, herein incorporatedby reference), BMP4 (see, Dalton, supra), GW788388(-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide)(see, e.g., Gellibert, et al., Journal of Medicinal Chemistry49(7):2210-2221 (2006)), SM16 (see, e.g., Suzuki, et al., CancerResearch 67(5):2351-2359 (2007)), IN-1130(3-(5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide)(see, e.g., Kim, et al., Xenobiotica 38(3):325-339 (2008)), GW6604(2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine) (see, e.g., deGouville, et al., Drug News Perspective 19(2):85-90 (2006)), SB-505124(2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride) (see, e.g., DaCosta, et al., Molecular Pharmacology65(3):744-752 (2004)) and pyrimidine derivatives (see, e.g., thoselisted in Stiefl, et al., WO2008/006583, herein incorporated byreference). Further, while “an ALK5 inhibitor” is not intended toencompass non-specific kinase inhibitors, an “ALK5 inhibitor” should beunderstood to encompass inhibitors that inhibit ALK4 and/or ALK7 inaddition to ALK5, such as, for example, SB-431542 (see, e.g., Inman, etal., J, Mol. Phamacol. 62(1): 65-74 (2002). Without intending to limitthe scope of the invention, it is believed that ALK5 inhibitors affectthe mesenchymal to epithelial conversion/transition (MET) process.TGFβ/activin pathway is a driver for epithelial to mesenchymaltransition (EMT). Therefore, inhibiting the TGFβ/activin pathway canfacilitate MET (i.e., reprogramming) process.

TGF beta receptor inhibitors can include antibodies to, dominantnegative variants of and siRNA or antisense nucleic acids that targetTGF beta receptors. Specific examples of inhibitors include but are notlimited to SU5416;2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride (SB-505124); lerdelimumb (CAT-152); metelimumab (CAT-192);GC-1008; ID11; AP-12009; AP-11014; LY550410; LY580276; LY364947;LY2109761; SB-505124; SB-431542; SD-208; SM16; NPC-30345; Ki26894;SB-203580; SD-093; Gleevec; 3,5,7,2′,4′-pentahydroxyflavone (Morin);activin-M108A; P144; soluble TBR2-Fc; and antisense transfected tumorcells that target TGF beta receptors. (See, e.g., Wrzesinski, et al.,Clinical Cancer Research 13(18):5262-5270 (2007); Kaminska, et al., ActaBiochimica Polonica 52(2):329-337 (2005); and Chang, et al., Frontiersin Bioscience 12:4393-4401 (2007).)

BMP Inhibitors

Exemplary BMP pathway inhibitors include, but are not limited to:Noggin, BMP receptor inhibitors, inhibitors of SMAD 1/5/8phosphorylation, inhibitors of the interaction of SMAD 1/5/8 and SMAD 4,and activators/agonists of SMAD 6 and SMAD 7. The categorizationsdescribed below are merely for organizational purposes and one of skillin the art would know that compounds can affect one or more pointswithin a pathway, and thus compounds may function in more than one ofthe defined categories.

Inhibitors of SMAD 1/5/8 phosphorylation include, but are not limitedto, antibodies to, dominant negative variants, antisense nucleic acids,and small molecules that target SMAD 1, SMAD 5, or SMAD 8. Specificexamples of inhibitors include LDN-193189 and Dorsomorphin (commerciallyavailable from, e.g., Stemgent).

Activators/agonists of SMAD 6 and SMAD 7 include, but are not limitedto, nucleic acids encoding SMAD 6 or SMAD 7 proteins or fragmentsthereof and polypeptides and fragments encoded by said nucleic acids.(See, e.g., Miyazono, et al., U.S. Pat. No. 6,534,476, incorporatedherein by reference.)

BMP receptor inhibitors include, but are not limited to, antibodies to,dominant negative variants of, siRNA or antisense nucleic acids, orsmall molecules that target BMP receptors. Specific examples ofinhibitors include, but are not limited to, DMH-1, Dorsomorphindihydrochloride, and LDN-193189 (commercially available, from, e.g.,Tocris Biosciences).

Hedgehog Pathway Inhibitors

Exemplary Hedgehog pathway inhibitors include, but are not limited to:inhibitors of Hedgehog binding to its receptor and inhibitors ofsignaling proteins downstream of Hedgehog (e.g., Patched or Smoothened).Specific examples of Hedgehog pathway inhibitors include cyclopamine,KAAD-Cyclopamine, SANT-1, SANT-2, U18666A, JK184, GANT 58, GANT 61, andGDC-0449 (commercially available from, e.g., Stemgent and TocrisBiosciences). Hedgehog pathway inhibitors are also described in Wu etal., WO 2006/050351, incorporated herein by reference. Additionalinhibitors include but are not limited to monoclonal or polyclonalantibodies or immunogenically active fragments thereof, peptideaptamers, a Hedgehog binding protein or protein that binds to asignaling protein downstream of Hedgehog, an antisense molecule to aHedgehog nucleic acid or a nucleic acid encoding signaling proteindownstream of Hedgehog, an RNA interference molecule, a morpholinooligonucleotide, a peptide nucleic acid (PNA), a ribozyme, and apeptide.

Notch Inhibitors

Exemplary Notch inhibitors include, but are not limited to: inhibitorsof Notch ligands, inhibitors of Notch activation, and inhibitors ofgamma-secretase. Specific examples of Notch pathway inhibitors includeDAPT, LY411575, L-685458, RO429097, MK-0752, MRK-003, and[11-endo]-N-(5,6,7,8,9,10-hexahydro-6,9-methanobenzo[a][8]annulen-11-yl)-thiophene-2-sulfonamide.(See, e.g., Fan et al., Cancer Res., 66:7445-52 (2006)). Additionalinhibitors include but are not limited to monoclonal or polyclonalantibodies (e.g., against Notch or against Notch ligands) orimmunogenically active fragments thereof, peptide aptamers, an antisensemolecule to Notch, an RNA interference molecule, a morpholinooligonucleotide, a peptide nucleic acid (PNA), a ribozyme, and apeptide.

D. Markers

Differentiation can be monitored by the expression of differentiationmarkers. Typical differentiation markers can be used forcharacterization of the cells of the invention. For example, cardiacdifferentiation markers include, e.g., early-stage cardiac markersBrachyury (T) and Mesp1; mid-stage cardiac markers Flk1, Nkx2.5, Isl1,and Gata-4; late-stage cardiac markers Mlc2a, myosin (sarcomeric andMHCb), and cTroponin T. Neural stem cells markers include Sox1 Pax6,PLZF, Sox2, and Dach1. Markers for differentiated neurons include Tuj1,MAP2, Dcx, Neurofilament, NenN, and Synapsin. Markers for subtypes ofdifferentiated neurons include TH, AchE, ChAT, Serotonin, VGluT, Nurr1,and GABA. Astrocyte (astroglial cells) markers include GFAP.Oligodendrocyte markers include CNPase, O1, and O4. Pancreaticdifferentiation markers include, e.g., early- or mid-stage pancreaticmarkers: Sox17, Foxa2, HNF4a, HNF1b, HNF6, Pax6, Sox9; and late-stagepancreatic markers: Pdx1, Nkx6.1, Nkx2.2, Isl1, MafA, c-peptide, andInsulin.

Methods known in the art can be used to characterize the cells of theinvention. Gene expression levels can be detected by, e.g., real-timePCR or real-time RT-PCR (e.g., to detect mRNA), and/or by western blotor other protein detection technique. Other cell differentiationcharacteristics such as cell morphologies associated with differentiatedstem cells can also be used for characterization of the cells of theinvention. For example, in some embodiments, the cell has a neuronmorphology, i.e., a round cell body having projections (axons/dendrites)greater than 2 times the diameter of the cell body. In some embodiments,the cell has a cardiomyocyte morphology, i.e., cells having crossstriations formed by alternating segments of actin and myosin.

E. Administration

Any of the differentiated cells (allogenic or autologous) as describedherein can be introduced into a subject. For example, the cells can bedelivered to a particular tissue or organ of a subject in need thereof,to ameliorate or treat a disease or condition.

In some embodiments, when cardiac cells (e.g., cardiomyocytes) aregenerated, the cells can be useful for generating artificial hearttissue, e.g., for implanting into a mammalian subject. In someembodiments, the method is useful for replacing damaged heart tissue(e.g., ischemic heart tissue) and/or for stimulating endogenous stemcells or non-cardiomyocyte somatic cells resident in the heart toundergo cardiomyogenesis. Where a subject method involves introducing(implanting) a cardiomyocyte into an individual, allogenic or autologoustransplantation can be carried out.

Individuals in need of treatment with cardiac cells (e.g.,cardiomyocytes) generated as described herein include, but are notlimited to, individuals having a congenital heart defect; individualssuffering from a condition that results in ischemic heart tissue, e.g.,individuals with coronary artery disease; and the like. A subject methodis useful to treat degenerative muscle disease, e.g., familialcardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy,restrictive cardiomyopathy, or coronary artery disease with resultantischemic cardiomyopathy.

Individuals in need of treatment with pancreatic cells generated asdescribed herein include, but are not limited to, individuals havingType I or Type II diabetes or who are pre-diabetic or insulin resistant.

Individuals in need of treatment with neural cells generated asdescribed herein include, but are not limited to, individuals havingnerve damage, paralysis, etc.

Individuals in need of treatment with retinal pigmented epithelium cellsgenerated as described herein include, but are not limited to,individuals having macular degeneration (e.g., age-related maculardegeneration), photoreceptor dystrophy, or merTK dystrophy.

For administration to a mammalian host, a cardiomyocyte, pancreatic, orneuronal (or other cell having a second cell fate as described herein)population generated using a subject method can be formulated as apharmaceutical composition. A pharmaceutical composition can be asterile aqueous or non-aqueous solution, suspension or emulsion, whichadditionally comprises a physiologically acceptable carrier (i.e., anon-toxic material that does not interfere with the activity of theactive ingredient). Any suitable carrier known to those of ordinaryskill in the art may be employed in a subject pharmaceuticalcomposition. The selection of a carrier will depend, in part, on thenature of the substance (i.e., cells or chemical compounds) beingadministered. Representative carriers include physiological salinesolutions, gelatin, water, alcohols, natural or synthetic oils,saccharide solutions, glycols, injectable organic esters such as ethyloleate or a combination of such materials. Optionally, a pharmaceuticalcomposition may additionally contain preservatives and/or otheradditives such as, for example, antimicrobial agents, anti-oxidants,chelating agents and/or inert gases, and/or other active ingredients.

In some embodiments, a population of cells having the second cell fateis encapsulated, according to known encapsulation technologies,including microencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883;4,353,888; and 5,084,350). Where the cell are encapsulated, in someembodiments the cells are encapsulated by macroencapsulation, asdescribed in, e.g., U.S. Pat. Nos. 5,284,761; 5,158,881; 4,976,859;4,968,733; 5,800,828 and published PCT patent application WO 95/05452.In some embodiments, a cell population having a second cell fate ispresent in a matrix.

The methods described herein can be applied in vitro (e.g., in culture)or partly or completely in vivo. In in vivo applications, in someembodiments, vectors, proteins and/or small molecules are targeted toone or more target cell in the body such that one or more reprogrammingfactors are increased in the target cells to generate a lessdifferentiated cell. Alternatively, such less differentiated cells canbe generated in vitro and then delivered to a target tissue or organ. Insome embodiments, the resulting less differentiated cell is present in atissue or organ and is induced to differentiate into the second cellfate due at least in part to endogenous signals from the tissue or organitself. Alternatively, the less differentiated cells can bedifferentiated into the cells having the second cell fate, for exampleas described herein, albeit in vivo.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Direct Reprogramming of Fibroblasts to a Cardiac Fate Abstract

Here we show that conventional reprogramming towardspluripotency—overexpresssing Oct4, Sox2, Klf4, and c-Myc—can be shortcutand directed towards cardiogenesis in a fast and efficient manner. Withas little as four days of transgene expression, mouse embryonicfibroblasts (MEFs) can be directly reprogrammed to spontaneouslycontracting patches of differentiated cardiomyocytes over a period of11-12 days. Several lines of evidence suggest that a pluripotentintermediate is not involved. Our method represents a unique strategythat allows a transient, plastic developmental state established earlyin reprogramming to effectively serve as a cellular transdifferentiationplatform, the utility of which likely extends beyond cardiogenesis. Ourstudy has potentially wide-ranging implications for iPSC factor-basedreprogramming and broadens the existing paradigm.

Introduction

The mammalian heart lacks significant regenerative capacity. In vitrogeneration of autologous cardiac cells for transplantation is a key areaof study for the effective treatment of heart disease. A recent reportof successful transdifferentiation of somatic cells to a cardiac fate invitro (Ieda, M. et al., Cell 142, 375-86 (2010)) has raised thepossibility that this process might eventually be used for cell-basedcardiac therapy. However, speed and efficiency must first be improved,especially if the ultimate goal is to provide a faster and saferalternative to the re-differentiation of autologous induced pluripotentstem cells (iPSCs) (Schenke-Layland, K. et al., Stem Cells 26, 1537-46(2008)).

We and others in the reprogramming field have often noted the very rarebut generally reproducible appearance of various terminallydifferentiated cell types—including spontaneously contracting cardiaccolonies—during the later stages of iPSC formation. In light of theseobservations, and because induced pluripotency is established in astep-wise and stochastic fashion (Hanna, J. et al., Nature 462, 595-601(2009); Stadtfeld, M. et al., Cell Stem Cell 2, 230-40 (2008)), wehypothesized that it might be possible to modify the reprogrammingprocess to favour such alternative outcomes. We reasoned that providingthe appropriate developmental cues after an initial epigenetic“activation phase” may allow us to hijack conventional reprogramming atthis early, unstable stage, and specifically shift the outcome towardscardiogenesis.

Results Three-Factor Reprogramming is Sufficient to Activate the EarlyCardiac Program

To test the validity of our hypothesis and gauge technical feasibility,we virally transduced MEFs harbouring a Nebulette-LacZ reporterexpressed only in nascent myocardium (Cai, C. L. et al., Development132, 2475-87 (2005)) with the four Yamanaka factors (Takahashi, K. andYamanaka, S., Cell 126, 663-76 (2006)) and colorimetrically monitoredearly cardiogenesis. We modified standard reprogramming medium byremoving leukaemia inhibitory factor (LIF) to avoid the generationand/or maintenance of pluripotent cells. We also tried differentbasement membranes (gelatin, fibronectin, Matrigel, or Geltrex) and theaddition of foetal bovine serum (FBS) at concentrations of 1-15% toincrease cell viability and/or the cardio-inductive properties of themedium. When cells were cultured on Matrigel or Geltrex in LIF-freemedium containing 5% FBS, we observed transient (≦2 days) butwide-spread (approx. 50%)β-galactosidase expression in nascent coloniesafter a week. Importantly, systematic omission of factors from the viralcocktail revealed that c-Myc was dispensable, i.e., three-factortransduction worked equally well (FIG. 1 a).

Induction of Spontaneous Contraction Requires Growth Factor Signallingand is Enhanced by a Small Molecule

Having successfully initiated the early cardiogenic program in MEFs, wenext sought to induce robust activation of mid-stage cardiac geneexpression and to ultimately generate spontaneously contracting patchesof cardiac cells, a hallmark of terminal differentiation that is easilyobserved and quantified. To this end, we treated three-factor transducedMEFs with chemically defined media (CDM) as well as cytokines and smallmolecules that might enhance cardiogenesis by regulating TGFβ, bonemorphogenetic protein (BMP), Hedgehog, Wnt, or Notch pathway activity.Treatments were done individually and in combination, and at differenttimes during the reprogramming process. Despite the generalunsuitability of the resulting protocol for iPSC generation, we alsoexplored whether small-molecule inhibition of JAK-STAT (Januskinase-signal transducer and activator of transcription), PI3K(phosphatidylinositol 3-kinase), or Wnt signalling during the earlystages of the protocol might boost the effectiveness of cardiomyogenesisby preventing even a small number of iPSC intermediates from forming.Finally, we experimented with a more gradual transition betweendifferent media by lowering FBS concentration step-wise in increments of1-4% over 3-6 days (immediately prior to switching to CDM). Afterseveral rounds of experimentation employing many different combinations,we determined the following conditions to be the most effective: cellsare first exposed to reprogramming media containing 15% FBS and 5%knockout serum replacer (KSR) for six days, followed by a switch to 1%FBS and 14% KSR for three days. During this initial nine-day period, thesmall-molecule JAK inhibitor JI1 is continuously kept in the media at aconcentration of 0.5 μM. From day nine onwards, the cells are culturedin CDM, with the cardioinductive growth factor BMP4 (Snyder, A. et al.,J Cell Biochem 93, 224-32 (2004)) being added at 20 ng/ml for the firstfive days (FIG. 1 b).

Using three-factor transduction with the above conditions, we observedrobust expression of the mid-stage cardiac markers (Martin-Puig, S. etal., Cell Stem Cell 2, 320-31 (2008)) Flk1, Nkx2.5, and Gata-4 startingon days 9-10 (FIG. 1 c). Late-stage markers including cardiac troponinT, sarcomeric myosin heavy chain, and α-actinin were observed from day11 onwards. Simultaneously, Connexin-43 staining became apparent alongmany cells' periphery. Interestingly, regardless of the time point atwhich ICC was performed, we only detected the atrial isoform of myosinlight chain (Mlc-2a), indicating that the cardiomyocytes being generatedwere mostly, if not exclusively, of the atrial subtype (FIG. 1 d).

The earliest wave-like spontaneous contractions also began on day 11,and many colonies were seen forcefully contracting in their entirety byday 15. Although not absolutely required, BMP4 appears to be the majordriving force behind the robust development of beating: on average, itsaddition increased the formation of contracting patches nearly 150-fold(149±13 patches per 100,000 MEFs plated, n=6). Strikingly, sequentialapplication of the JAK inhibitor JI1 and BMP4 can even further increasethe number of contracting patches: we have obtained as many as 317independently contracting patches per 100,000 MEFs plated by day 21(mean=257±17, n=6; FIG. 2 a). Under these conditions, the incidence ofbeating in terms of total colony number could be as high as 90%(mean=79±4%, n=5; FIG. 2 b). The dramatic nature of this shift and thepervasiveness of the cardiogenic outcome were especially evident inwhole-well microscopic analyses, in which all large colonies in a wellcould be seen expressing high levels of cTnT and spontaneouslycontracting by day 18. Conversely, they did not express above-backgroundlevels of Nanog-GFP at the same time point (FIG. 2 c).

JI1 was most effective when applied continuously for the first ninedays—the period during which there might be a remote possibility ofgenerating iPSC intermediates due to the use of Yamanaka factors (FIG. 1b). Accordingly, its effectiveness declines with shorter treatmentperiods; however, extending JI1 treatment beyond nine days to overlapwith CDM and BMP4 application also appears to be counterproductive (datanot shown). This latter observation is supported by a recently reportedrequirement for JAK-STAT signalling in cardiomyogenesis (Snyder, M. etal., J Biol Chem 285, 23639-46 (2010)). In the case of BMP4, five daysof treatment was optimal (FIG. 1 b); both shorter and longer periods oftreatment proved detrimental to the development of beating cardiaccolonies (data not shown). While its precise role in direct cardiacdifferentiation remains to be clarified, BMP4 is most likely drivingcardiac induction from nascent precursors during a criticaldevelopmental window (Cohen, E. D. et al., Development 135, 789-98(2008); Klaus, A. and Birchmeier, W., Pediatr Cardiol 30, 609-16(2009)). It could further be speculated that the cardioinductive effectof JI1 treatment might result from an indirect expansion of thisprecursor pool: by preventing cells from becoming primed for inducedpluripotency, a larger number may ultimately become available for BMP4to act upon and prime for cardiogenesis instead.

Transdifferentiation Yields a Substantial Number of HighlyDifferentiated and Functionally Responsive Cardiomyocytes

Although contracting colony number serves as a very convenient metricfor inter-sample comparisons of reprogramming success and efficiency, itdoes not allow for a quantification of the individual cardiomyocyteyield. To this end, we utilized fluorescence-activated cell sorting(FACS) on days 0, 10, and 18, examining both precursor (Isl1⁺, Nkx2.5⁺)and more mature (cTnT⁺) cardiac populations. A modest increase wasobserved for all three markers by day 10, with significant increases byday 18. Isl1⁺ and Nkx2.5⁺ cells did not increase to the same extent ascTnT⁺ cells, presumably because the former are more strongly expressedduring the earlier stages of cardiac development (Qyang, Y. et al., CellStem Cell 1, 165-79 (2007)). Remarkably, nearly 40% (39±2%, n=5) ofcells had become cTnT⁺ at the later time point (FIG. 2 d). Based onthese data, the final yield of cTroponin T⁺ cells was conservativelyestimated to be 120,000 per 100,000 MEFs plated or 1.2 cardiomyocytesper fibroblast (100,000 starting MEFs reproducibly translated into a day18 harvest of approximately 300,000-350,000 total cells).

To more closely examine the degree of differentiation among thepopulation of contracting cells, we examined their beating frequency,calcium flux patterns, and response to chronotropic agents. Contractionfrequency of cardiomyocytes on day 18 varied from a minimum of 4 beatsper minute (BPMs) (data not shown) to a maximum of 130 BPMs. While thispattern implied a wide range of development/differentiation, the latterobservation suggested that at least a subset of cardiomyocytes wereacquiring highly differentiated traits (Kuzmenkin, A. et al., FASEB J23, 4168-80 (2009)). Accordingly, many contracting patches exhibitedcharacteristic calcium transients (Ieda, M. et al., Cell 142, 375-86(2010); Shah, A. P. et al., Cardiovasc Res 87, 683-93 (2010)) (FIG. 3 a)(Stieber, J. et al., Proc Natl Acad Sci USA 100, 15235-40 (2003)), thefrequency of which could be reversibly modulated with 1 μM isoproterenolor 10 μM carbachol, indicating proper responsiveness to β-adrenergic andmuscarinic signalling, respectively (Kuzmenkin, A. et al., FASEB J 23,4168-80 (2009)) (FIG. 3 b). Addition of isoproterenol significantlyincreased the frequency of spontaneous calcium transients and shortenedthe decay period (τ), while carbachol had the opposite effects (FIG. 3c). Total contracting colony number consistently levelled off beyond day18, but most colonies continued to contract well beyond the end point ofour assays (day 21), with some patches still contracting after fiveweeks (data not shown).

Next, we enzymatically dissociated contracting patches into individualcardiomyocytes and carried out electrophysiological measurements (FIG. 3d). The spontaneous action potentials (APs) generated in these cellsclosely resembled “atrial-like” (and to a lesser extent“pacemaker-like”) APs reported elsewhere (Stieber, J. et al., Proc NatlAcad Sci USA 100, 15235-40 (2003)), with a mean diastolic potential(MDP) of −61.0±1.2 mV and a mean overshoot of 13.9±4.9 mV (n=3). Assuch, these data corroborate immunocytochemistry results suggesting amostly atrial phenotype for the cardiomyocytes being generated (FIG. 1d).

Of note, we have repeatedly observed a small number of contractingsingle cells exhibiting characteristic cardiac troponin T staining evenprior to enzymatic dissociation, indicating that nascent cardiomyocytescan differentiate, and perhaps even arise, outside of the context or“niche” of a developing colony (FIG. 1 e). Such a phenomenon wouldsuggest that a subset of cells might be taking a more directtransdifferentiation path (i.e., perhaps not having to rely onextracellular cues from neighbouring cells in colony), a hypothesisconsistent with the stochastic nature of Yamanaka factor-inducedreprogramming.

Next, we conducted chromatin immunoprecipitation (ChIP) assays toascertain the nature and extent of certain epigenetic changes takingplace at four loci: Oct4, Actn2 (encoding α-actinin), Ryr2 (encoding thecardiac ryanodine receptor), and Tnnt2 (encoding cardiac Troponin T).Relative levels of trimethylation at histone H3 lysine 4 (H3K4me3) andlysine 27 (H3K27me3) in the promoter regions of these genes wereexamined. As expected, due to strong exogenous expression of theYamanaka factors during the initial part of the protocol, we observed aninitial burst of activating H3K4me3 (with a concomitant decrease inH3K27 m3) at the Oct4 promoter by day 10, followed by a return to theH3K27me3-dominated, repressed state seen in the starting MEFs by day 19(FIG. 4 a). The promoters of the three cardiac genes, all of which areexpressed at the later stages of cardiomyogenesis, also exhibitedenrichment in H3K4me3 by day 10, but unlike Oct4, this trend did notstop or reverse; by day 19, H3K4me3 enrichment was extensive compared tothe starting MEFs. Strikingly, it even exceeded levels found incardiomyocytes derived from mESCs (FIG. 4 a). Concurrently, levels ofrepressive H3K27me3 at the cardiac loci had fallen far below thestarting fibroblasts' levels, and were approaching those of the controlcardiomyocytes.

Transdifferentiated Cardiomyocytes Most Likely do not Arise fromContaminating Cardiac Precursors or iPSC Intermediates

To rule out the possibility that beating patches might be arising fromrare multipotent cardiac precursor cells in our MEF cultures, we testedour method on tail-tip fibroblasts (TTFs), a much more homogenous sourceof fibroblasts that does not contain any cardiac cells. As postnatal andadult cells are more refractory to reprogramming (Markoulaki, S. et al.,Nat Biotechnol 27, 169-71 (2009)), it was necessary to use all fourreprogramming factors, including c-Myc, to induce cardiac colonyformation. Surprisingly, on day 12—only a day later than when MEFs wereused—we observed a small number of colonies beginning to contract.Despite this striking similarity in timing, the number of beatingpatches generated per 100,000 cells plated (115±7, n=6) was lower thanthe yield from MEFs reprogrammed under the same conditions (145±6, n=6;p<0.01).

The conditions utilized in our protocol are not conducive to theestablishment or maintenance of iPSCs (Blelloch, R. et al., M., CellStem Cell 1, 245-7 (2007)). We do not utilize feeder cells or LIF, andswitch to chemically defined media containing cardiogenic moleculesearly in the protocol. Most importantly, even with three-factortransduction, the first contracting patches emerge within 12 days, amuch shorter period than the approximately three-week minimum requiredto generate iPSCs with three factors (Nakagawa, M. et al., NatBiotechnol 26, 101-6 (2008); Wernig, M. et al., Cell Stem Cell 2, 10-2(2008)). Strikingly, cardiac reprogramming of TTFs also proceeds at thesame pace, even though such postnatally derived cells would normally beexpected to reprogram more slowly (Okada, M. et al., Biochim BiophysActa 1800, 956-63 (2010)). Despite these considerations, we sought tomore rigorously rule out the possibility that our method might depend onthe generation of a transient pluripotent intermediate, there-differentiation of which could conceivably give rise tocardiomyocytes.

First, we reasoned that if generation of iPSCs prior to the switch toCDM were critical, using standard reprogramming media with LIF for thefirst nine days should yield a greater number of contracting colonies.However, this substitution actually resulted in a four-day delay in theonset of beating and a ˜25-fold decline in beating colony number (FIG. 4b). A critical implication of this result is that early reprogrammingevents—brought about primarily by the overexpression of the Yamanakafactors—appear to be generating a heterogeneous population of cells withdiverse developmental potential, as even standard reprogramming mediaallows for the generation of a very small number of cardiac progenitors.Further, conditions that favour the development of iPSCs must do so atthe expense of other potential outcomes of the reprogramming process:although marginally permissive, standard reprogramming media is a verypoor choice for the initiation of direct cardiac reprogramming.

Prolonged Induction of the Pluripotency Program InhibitsTransdifferentiation

Second, the rapid induction of beating suggests that lineage commitmentdecisions are made quite early. We speculate that the reprogrammingfactors (especially Oct4) mostly function to “erase” cell identity byepigenetic mechanisms, and do not directly activate lineage-specificgenes. Conversely, exposure to external, cardiac-specific signals (inthis case BMP4 treatment) likely has role(s) in early lineagespecification and/or the induction of terminal differentiation. In anycase, overexpression of reprogramming factors should be only transientlyrequired to allow for activation of lineage-specific gene networks, withprolonged expression likely to be detrimental. To test this hypothesis,we generated secondary MEFs harbouring doxycycline-inducible transgenes(Wernig, M. et al., Nat Biotechnol 26, 916-24 (2008)) and a Nanog-GFPreporter (Hanna, J. et al., Cell 133, 250-64 (2008)) to monitorestablishment of pluripotency (Silva, J. et al., Cell 138, 722-37(2009)). Strikingly, a mere four days of doxycycline treatment wassufficient to induce beating on day 11. The optimal duration of drugtreatment was between five and six days, with treatment longer than ninedays not producing any contracting cardiac cells at all (FIG. 4 d).Conversely, generation of iPSC colonies using this particular systemrequires a minimum of 12 days of transgene induction (Wernig, M. et al.,Nat Biotechnol 26, 916-24 (2008); Brambrink, T. et al., Cell Stem Cell2, 151-9 (2008)). Even the shortest transgene expression requirementreported to date is seven to eight days (Stadtfeld, M. et al., NatMethods 7, 53-5 (2010)). In short, optimal cardiac reprogrammingrequires transgenes to be inactivated well before pluripotency can beendogenously established.

We also tested our protocol on mESCs to establish whether it had acardiogenic effect on pluripotent cells. Substituting mESCs for day 0,3, 6, or 9 cultures in our protocol did not produce any contractingpatches by day 21 or beyond, indicating that our protocol could notbring about the development of cardiac colonies from iPSCs, even if theywere arising during the direct reprogramming process (FIG. 4 c). Insummary, these results demonstrate the establishment of bona fidepluripotency during our protocol would in fact constitute a strongbarrier to successful cardiac reprogramming.

Cardiac Transdifferentiation and iPSC Generation Represent Parallel,Non-Overlapping Processes

Third, quantitative RT-PCR results show that the cardiogenic program isalready underway by days 4-6, as indicated by the robust expression ofMesp1 and GATA4 transcripts (FIG. 5 a). We did note that the randomnessand asynchrony inherent in reprogramming perhaps resulted in aheterogeneous population of cardiac mesoderm cells and, in turn, higherinter-sample variation. Conversely, levels of reprogramming factortranscripts decrease precipitously following doxycycline withdrawal andgenerally remain very low. Endogenous Nanog transcripts are notdetectable until days 8-9, and at very low levels (FIG. 5 b).Consequently, most colonies do not express any Nanog-GFP at all; only asmall subset show levels of fluorescence scarcely above background,starting on days 9-12 (data not shown). Thus, any low-level activationof Nanog coincides with early expression of mature cardiac markers suchas the 0 isoform of myosin heavy chain (MHCβ) and the initiation ofspontaneous contraction on day 11 (FIG. 5 a). Although this concurrentactivation of late-stage iPSC and cardiomyocyte markers effectivelyrules out the possibility that the former could somehow give rise to thelatter, we sought to definitively show that the development ofspontaneous contraction does not require activation of the endogenouspluripotency network. To accomplish this, we took daily photographs oflarge numbers of cells undergoing direct reprogramming andretrospectively evaluated Nanog-GFP expression in colonies thateventually began contracting. Not surprisingly, we found that thesecolonies had not expressed even minute amounts of Nanog-GFP at any timeduring their formation or differentiation (FIG. 5 c).

FACS analyses of SSEA-1⁺ and Nanog⁺ cells on days 0, 10, and 18corroborated the above results: even on day 18, Nanog⁺ cells onlycomprised 1% of the population. SSEA-1⁺ cells made up a similarly smallfraction of the cells on day 18. On day 10, these latter cellsrepresented close to 10% of the population (FIG. 2 d). However, sinceSSEA-1 is one of the earliest markers induced upon Yamanaka factoroverexpression (Stadtfeld, M. et al., Cell Stem Cell 2, 230-40(2008))—and is in fact expressed in a range of differentiated precursorcells (Anjos-Afonso, F. and Bonnet, D., Blood 109, 1298-306 (2007);Koso, H. et al., Dev Biol 292, 265-76 (2006))—it is not a marker of bonafide pluripotency. This notion is especially valid given the lack ofconcomitant Nanog expression (Silva, J. et al., Cell 138, 722-37 (2009))and the precipitous decline of the SSEA-1⁺ population by day 18. Wesurmise that the higher levels on day 10 may result from a delayeddownregulation of exogenously induced SSEA-1 following dox withdrawal.

Taken together, these results indicate that any iPSC-like cellsgenerated in our protocol are a minor by-product and most likely do notcontribute to cardiomyocyte formation. The observation that JAK-STATpathway inhibition by JI1 can shift the balance considerably in favourof cardiomyogenesis constitutes strong evidence that the relationshipbetween iPSC and cardiomyocyte formation is zero-sum in nature, i.e.,that they represent mutually exclusive outcomes.

Discussion

We have shown that brief reactivation of reprogramming factors inembryonic and adult fibroblasts can be used to rapidly generatecontracting cardiomyocytes—almost certainly without going through apluripotent intermediate. Compared to transdifferentiation bylineage-specific factor overexpression, our protocol is nearly threetimes as fast: the first spontaneous contractions begin after 11 daysvs. 4-5 weeks (Ieda, M. et al., Cell 142, 375-86 (2010)). Moreover, theefficiency in terms of cTnT⁺ cardiomyocyte yield is several fold higher:we have obtained approximately 1.2 cTnT⁺ cells for each fibroblastplated (vs. an estimated maximum of 0.2) (Ieda, M. et al., Cell 142,375-86 (2010)). This latter feat is most likely made possible by thegeneration of mitotically active precursor cells akin tomultipotent/s/1⁺ cardiovascular progenitors (MICPs) (Qyang, Y. et al.,Cell Stem Cell 1, 165-79 (2007)), as suggested by gene expression data.It is tempting to speculate that these intermediate cells, ifsuccessfully isolated and stabilized in culture, could eventually becomean expandable and renewable source for not just cardiomyocytes, but manyother terminally differentiated cardiovascular cells as well.

The process we have discovered bears a striking resemblance toregeneration blastema formation in zebrafish and frogs, during which thestage is set for transdifferentiation by transient low-level expressionof pluripotency factors (esp. Oct4 and Sox2) rather than are-establishment of pluripotency (Christen, B. et al., BMC Biol 8, 5(2010)). While this suggests that the two processes could share a commonmechanism, confirmation of this hypothesis will require a more detailedunderstanding of the molecular underpinnings of reprogramming and itsvarious intermediate stages (Stadtfeld, M. et al., Cell Stem Cell 2,230-40 (2008)). The key role here for reprogramming factors is likelythe induction of a developmentally more naïve, open-chromatin statemarked by high epigenetic instability (Meshorer, E. et al., Dev Cell 10,105-16 (2006); Hochedlinger, K. and Plath, K., Development 136, 509-23(2009); Artyomov, M. N. et al., PLoS Comput Biol 6, e1000785). Highlyunstable intermediate populations may give rise to a multitude of celltypes as they rapidly “relax” back into epigenetically more stablestates, among which pluripotency is one of many possible outcomes (FIG.6). This model underscores the unique versatility and potential of ourtransdifferentiation scheme as compared to lineage-specifictranscription factor ovexpression, namely the likelihood that precursorsand/or fully differentiated cell types from many different lineagescould be derived simply by using different inductive signals. Thus, forthe generation of autologous tissue, transdifferentiation offers apotentially very attractive alternative to the rather circuitous iPSCmethodology.

Material and Methods

Cell culture and media: All MEFs (129S2/SvPasCrlf) were derivedaccording to WiCell protocols(https://www.wicell.org/index.php?option=com_docman&task=doc_download&gid=687).TTFs were a kind gift of Dr. Hans Schöler. Secondary MEFs were derivedas previously described (Wernig, M. et al., Nat Biotechnol 26, 916-24(2008)). All results were initially obtained and confirmed with theNebulette-LacZ MEFs; to ensure reproducibility and minimizeinter-experimental variation, reported results almost exclusively derivefrom experiments utilizing secondary dox-inducible MEFs using theoptimum induction period. MEFs were initially passaged on gelatin-coated(0.1% for 2 hours at 37° C.) tissue culture dishes in DMEM supplementedwith 10% FBS, 2 mM Glutamax and 0.1 mM NEAA (all components fromInvitrogen, Carlsbad, Calif.). Prior to viral transduction, cells wereseeded onto Matrigel (BD Biosciences, Franklin Lakes, N.J.) or Geltrex(Invitrogen)-coated plates (1:40, overnight at 4° C.) at 3.5×10⁴ cellsper well of a six well plate (7×10⁴ cells for TTFs) in the same media.12-24 hours after the addition of virus, cells were washed with PBS andswitched to reprogramming media: knockout DMEM with 1-15% knockout serumreplacement, 1-15% ES-qualified FBS, 1% Glutamax, 1% nonessential aminoacids, 0.1 mM β-mercaptoethanol, and 1% ESC-qualified nucleosides (allcomponents from Invitrogen except nucleosides, Millipore, Billerica,Mass.). When required for a control experiment, LIF (Millipore) was alsoadded at 10³ units ml⁻¹. At the indicated times, cells were again washedwith PBS and switched to CDM: RPMI-1640 supplemented with 0.5×N2, 1× B27(without vitamin A), 0.05% BSA fraction V, 0.5% Glutamax, and 0.1 mMβ-mercaptoethanol (all components from Invitrogen). BMP4 (Stemgent, SanDiego, Calif.) and/or JI1 (EMD, San Diego, Calif.) were added atmultiple timepoints for different durations. Fresh media was added atleast once every 48 hours throughout all experiments. For the induciblereprogramming of secondary MEFs, 2 μg/ml dox was used, with theexception of four-day induction experiments, where the first two days oftreatment were done at 4 μg/ml.

Retroviral packaging and transduction: Retroviruses encoding Oct4, Sox2,Klf4, and c-Myc were individually packaged in PLAT-E cells usingpMXs-based vectors (Addgene, Cambridge, Mass.) and infections werecarried out as previously described (Takahashi, K. et al., Nat Protoc 2,3081-9 (2007)).

LacZ assays: Nebulette-LacZ expression was detected in situ using abeta-galactosidase staining kit (Stratagene, San Diego, Calif.).

Immunocytochemistry and fluorescence microscopy: Cells were fixed in 4%paraformaldehyde (Sigma-Aldrich, St. Louis, Mo.) for 15 minutes, washedthree times with PBS (phosphate-buffered saline), and incubated in PBScontaining 0.3% Triton X-100 (Sigma-Aldrich) and 5% donkey serum(Jackson ImmunoResearch, West Grove, Pa.) for 1 hour at roomtemperature. All primary antibody incubations were done overnight at 4°C.: cardiac troponin T (CT3, Developmental Studies Hybridoma Bank, IowaCity, Iowa; 1:1,000); Flk1 (AF-644, R&D Systems; Minneapolis, Minn.;1:100); Gata4 (sc-25310, Santa Cruz Biotechnology, Santa Cruz, Calif.;1:200); Myosin heavy chain (MF20, Developmental Studies Hybridoma Bank,1:200); Nkx2.5 (sc-8697, Santa Cruz, 1:200); Mlc2v (aka My17; sc-34488,Santa Cruz, 1:50); Connexin-43 (610061, BD Biosciences, 1:100), andα-actinin (NBP1-40428, Novus, Littleton, Colo.; 1:100). Following threePBS washes, cells were incubated with the appropriate AlexaFluor-conjugated secondary antibodies (Invitrogen) for 1 hour at roomtemperature and nuclei were stained with DAPI (Sigma-Aldrich). Imageswere captured using a Zeiss AX10 microscope equipped with an Axiocam HRmcamera and processed using Axiovision 4.7.1 software. Colony trackingexperiments and whole-well microscopic imaging were done on a Pathway435 system (BD Biosciences).

Quantitative PCR: For each sample/timepoint, total RNA from two wells ofa six-well plate was extracted using the RNeasy Plus Mini Kit withQiashredder columns (Qiagen, Germany), after which 1 μg of RNA wasreverse transcribed with the iScript cDNA Synthesis Kit (Bio-Rad,Hercules, Calif.). All quantitative PCR reactions were done in duplicateon a CFX96 system with iQ SYBR Green Supermix (Bio-Rad), using onetwentieth of a cDNA reaction per replicate. Expression data werenormalized relative to ribosomal protein L7 transcript levels. Each setof reactions was repeated using cDNA from at least three independentexperiments. Primer sequences and details of amplification conditionsare available upon request.

Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR): ChIP wasperformed using a commercially available Magna ChIP G kit (Millipore).Briefly, histones and DNA were cross-linked by incubation in 1%formaldehyde. The chromatin was then sonicated to an average DNAfragment length of 200 to 500 bp. Equal amounts of soluble chromatinwere incubated with and without anti-normal rabbit IgG,anti-trimethyl-histone H3 lysine 4 (#17-614, Millipore; 3 ul/reaction)or anti-trimethyl-histone H3 lysine 27 (#17-622, Millipore; 4ug/reaction) that had been pre-incubated with secondary antibodiesconjugated to magnetic beads. After overnight antibody incubations,DNA-histone cross-linking was reversed and DNA fragments were purified.DNA fragments obtained without antibody were used as the input controls,whereas DNA fragments obtained with normal rabbit IgG were applied asnegative controls. One microliter of the resulting DNA solution fromeach condition was subject to real-time PCR reaction. Error barsrepresent standard deviation. Primer sequences used are available uponrequest.

Flow cytometry: Adherent cells on plates were washed with PBS anddissociated with trypsin (Invitrogen), Accutase (Innovative CellTechnologies, San Diego, Calif.), or collagenase II (Invitrogen) for10-45 minutes at 37° C. Cells were washed twice with ice-cold stainingbuffer (HBSS [Invitrogen] with 10 mM HEPES [Sigma], 2% FBS [Invitrogen],and 0.1% azide [Sigma]) and put through single-cell strainers (BDBiosciences) both times. Incubation with antibodies was done at 4° C.for 30 minutes at the volume and concentrations suggested by themanufacturer. Cells were washed three times with staining buffer andfixed in high-grade 4% paraformaldehyde (Electron Microscopy Sciences,Hatfield, Pa.) for 15 minutes at 4° C. Cells were washed again withstaining buffer and counted using a FACSCalibur flow cytometer andCellQuest software (BD Biosciences). Final analysis was later done withFlowJo software (Tree Star, Ashland, Oreg.). Antibodies used were asfollows: APC-conjugated anti-Flk1 (560070, BD Biosciences),PE-conjugated anti-SSEA1 (560142, BD Biosciences), Alexa647-conjugatedanti-Nanog (560279, BD Biosciences), anti-cTnT (ab10214, Abcam,Cambridge, Mass.), and anti-Nkx2.5 (sc-8697×, Santa Cruz). Unlabelledantibodies were conjugated to PE or APC using the appropriate LYNX rapidconjugation kit (AbD Serotec, Germany). All experiments included theappropriate isotype controls, conjugated using the same kits as abovewhen necessary.

Calcium imaging and testing of chronotropic agents: Cells were loadedwith Fluo-4-AM (5 μM, 30 min, 37° C.) (F14201, Invitrogen), and imagedwith an Olympus Fluoview 1000 inverted confocal microscope with a 40×oil immersion lens (numerical aperture 1.3). Line scan imaging mode wasused to measure spontaneous Ca2⁺ transients at 37° C. Image processingand data analysis were performed as previously described (Ouyang, K. etal., J Biol Chem 280, 15898-902 (2005)). For drug testing, allmeasurements were repeated 10 minutes after the addition ofisoproterenol (1 μM; Sigma) or Carbachol (10 μM; Sigma-Aldrich).

Electrophysiology: Contracting cell clusters were dissociated at 37° C.for 30-45 minutes using 1 mg/ml Collagenase type II in RPMI-1640 mediasupplemented with 10 mM HEPES and 5 mM sodium pyruvate (all reagentsfrom Invitrogen). Following repeated pipetting to ensure completedissociation, cells were allowed to adhere to fibronectin-coated Biocoatcoverslips (BD Biosciences) overnight in Cor.At media (Lonza, Germany).Single-cell patch clamp recordings were performed using an Axopatch 200Bamplifier and pClamp 10.0 software (HEKA Elektronik, Germany), asdescribed previously (Ouyang, K. et al., J Biol Chem 280, 15898-902(2005)). Spontaneous action potentials were recorded under whole-cellcurrent-clamp conditions with a patch pipette resistance of 4-6 MΩ.Standard external solution contained (in mM): NaCl 150, KCl 5.0, CaCl22.0, MgCl 2 1.0, HEPES 10, and glucose 10 (pH 7.4 adjusted with NaOH).Intracellular pipette solution contained (in mM): 150 KCl, 5.0 NaCl, 1.0MgCl2, 2.0 EGTA, 1.0 MgATP, 10 HEPES (pH 7.2 adjusted with KOH). Allexperiments were performed at room temperature (20-22° C.).

Videos: All videos were recorded using a Canon A65015 digital cameracustom-mounted to a Zeiss Axiovert 40C microscope.

Statistics: P values for the purpose of group comparisons werecalculated using Student's t test. Differences where p<0.05 wereregarded as significant.

Example 2 Direct Reprogramming of Neural Precursor Cells to a CardiacFate

Neural precursor cells (NPCs) transduced with four factors (Oct4, Sox2,Klf4, and c-Myc) can give rise to a small number of beating colonieswithin 18 days. On average, six beating colonies/50 k cells plated wereobtained when they were switched directly into reprogramming mediafollowing viral transduction (i.e. without overnight incubation in NPCmedia). Keeping the cells in NPC media for the first 24 hours aftertransduction only resulted in half the number of colonies, and they beatmore slowly. Interestingly, NPCs transduced with three factors only(Oct4, Sox2, and Klf4) gave rise to many pink colonies, presumablyhematopoietic blood islands.

Example 3 Direct Reprogramming of Fibroblasts to a Neural Fate

The simple yet powerful technique of induced pluripotency may eventuallysupply a wide range of differentiated cells for cell therapy and drugdevelopment. However, making the appropriate cells via inducedpluripotent stem cells (iPSCs) requires reprogramming of somatic cellsand subsequent re-differentiation. Given how arduous and lengthy thisprocess can be, we sought to determine whether it might be possible toconvert somatic cells into lineage-specific stem/progenitor cells ofanother germ layer in one step, bypassing the intermediate pluripotentstage. Here we show that transient induction of the four reprogrammingfactors (Oct4, Sox2, Klf4, and c-Myc) can efficiently transdifferentiatefibroblasts into functional neural stem/progenitor cells (NPCs) withappropriate signalling inputs. Compared to “iN cells” (induced neuronalcells), transdifferentiated NPCs have the distinct advantage of beingexpandable in vitro and retaining the ability to give rise to multipleneuronal subtypes and glial cells. Our results provide a new paradigmfor iPSCfactor-based reprogramming by demonstrating that it can bereadily modified to serve as a platform for transdifferentiation.

Introduction

Although successful transdifferentiation from one cell type to anotherby overexpressing lineage-specific genes in vivo (Takeuchi, J. K. etal., Nature 459(7247):708-711 (2009), Zhou, Q. et al., Nature455(7213):627-632 (2008)) and in vitro (Graf, T. et al., Nature462(7273):587-594 (2009); Ieda M. et al., Cell 142(3):375-386 (2010))has been reported, these methods are typically only effective whendevelopmentally closely related cell types are used. While thegeneration of iN cells (Vierbuchen, T. et al., Nature463(7284):1035-1041 (2010)) using neural-specific transcription factorshas established that inter-lineage transdifferentiation is also possiblein vitro, all transdifferentiation schemes to date entail overexpressionof different sets of lineage-specific transcription factors.Intriguingly, a recent study reporting single-factortransdifferentiation of fibroblasts into blood precursors using OCT4(Szabo, E. et al., Nature 468(7323):521-526 (2010)) is no exception: byvirtue of its long-term ectopic expression and extensive binding to theregulatory regions of key hematopoietic genes, OCT4 also appears to beparticipating in regulating hematopoietic programs acting as alineage-specific transcription factor in this context. The exceptionalaspect of this study is rather the ability to generate a mitoticallyactive progenitor population that can be further differentiated into avariety of blood cells a critical feat that has yet to be accomplishedin transdifferentiation to cardiac and neural lineages.

In an effort to devise a more general transdifferentiation strategy thatmight give rise to a broad array of unrelated cell types—includinglineage-specific precursors—we attempted to direct the conventional fouriPSC-factor-based reprogramming (Takahashi, K., et al., Cell126(4):663-676 (2006); Takahashi K. et al., Cell 131(5):861-872 (2007))toward alternative outcomes. Specifically, studies indicating that iPSCsare generated in a sequential and stochastic manner (Stadtfeld, M. etal., Cell Stem Cell 2(3):230-240 (2008); Brambrink, T. et al., Cell StemCell 2(2):151-159 (2008); Hanna J. et al., Nature (2009)) led us tohypothesize that we might be able to manipulate cells at an early andepigenetically highly unstable state induced by the reprogrammingfactors. Different conditions could potentially give rise to a multitudeof cell types (Artyomov, M. N. et al., PLoS Comput Biol 6(5):e1000785(2010)) with more stable epigenetic profiles. In this context, inducedpluripotency is only one—and perhaps among the less likely—of manypossible outcomes. Indeed, studies have found partially or incompletelyreprogrammed cells expressing multiple lineage-specific markers(Takahashi, K., et al., Cell 126(4):663-676 (2006); Mikkelsen, T. S. etal., Nature 454(7200):49-55 (2008); Meissner, A. et al., Nat Biotechnol25(10):1177-1181 (2007); Silva, J. et al., PLoS Biol 6(10):e253 (2008);Maherali, N. et al., Cell Stem Cell 1(1):55-70 (2007); Sridharan, R. etal., Cell 136(2):364-377 (2009)), although these cells did not appear torepresent physiologically relevant cell types. Accordingly, wehypothesized that it might be possible to deliberately bias the earlyreprogramming process toward a defined precursor cell type by employinginductive and/or permissive signalling conditions, after which thedesired cells could be selected and expanded.

In the present study, we have directly reprogrammed fibroblasts tofunctional neural stem/progenitor cells (NPCs) over an abbreviatedperiod of four-factor induction. This direct reprogramming process isclearly distinct from conventional reprogramming to iPSCs or forwarddifferentiation of pluripotent cells. Our findings not only representthe first successful transdifferentiation of somatic cells intoproliferating NPCs, but also form the basis of a new methodology forinter-lineage transdifferentiation into multi- or oligopotent cells.

Results

To rigorously test our hypothesis, we attempted an inter-lineagetransdifferentiation from fibroblasts to NPCs using the doxycycline(dox)-inducible secondary MEF system (Hanna J. et al., Nature (2009);Wernig, M. et al., Nat Biotechnol 26(8):916-924 (2008); Hanna, J. etal., Cell 133(2):250-264 (2008)). Inducible overexpression allowsprecise temporal control over the expression of the conventionaliPSC-reprogramming factors, avoiding potentially detrimental effectsarising from their constitutive overexpression. To ensure the survivalof MEFs during the beginning of the reprogramming procedure, they werekept in MEF and reprogramming initiation medium (RepM-Ini; without LIF)for the first 3-6 days of dox treatment. Thereafter, neuralreprogramming medium (RepM-Neural) was applied to induce the generationand/or proliferation of nascent NPCs. RepM-Neural contains FGF2, EGF andFGF4 to support NPCs (Ying, Q. L. et al., Nat Biotechnol 21(2):183-186(2003); Hitoshi, S. et al., Genes Dev 18(15):1806-1811 (2004)). We trieddox treatments between three and six days to determine the optimalduration of reprogramming factors expression (FIG. 7A). We initiallyfound that a minimum of three days of dox treatment was sufficient toobtain Pax6-positive colonies after additional 8-9 days in culture inRepM-Neural (FIG. 7B). These colonies typically contained severalhundred cells that nearly homogenously expressed PLZF, a rosette NSCmarker (Elkabetz et al., Genes Dev., 22(2):152-165 (2008)), and Pax6, anearly neural transcription factor (Walther, C and Gruss, P, Development,113(4), 1435-1449 (1991) (FIG. 7B,C,D). Extending dox treatment to sixdays increased the number of colonies (0.59%±0.01, n=2, colonygeneration efficiency). Surprisingly, we found that almost 100% ofcolonies showed neural transdifferentation regardless of the testedduration of dox treatment (95.9%±4.6, n=3, FIG. 7C).

We found typical neural rosette structures, PLZF expression, and luminalexpression of ZO-1 (Elkabetz, Y. et al., Genes Dev 22(2):152-165 (2008))in the transdifferentiated colonies (FIG. 7D). In many colonies, we alsofound Tuj1-positive neurons, some of which co-express the early corticalneuronal marker Dcx, and even the dopaminergic neuronal markertyrosine-hydroxylase (FIG. 7D). Importantly, these expression profilesare not observed in iN cells (Vierbuchen, T. et al., Nature463(7284):1035-1041 (2010)). Flow cytometry analysis of thetransdifferentiated cells revealed that a population of cells expressingProminin-1, PSA-NCAM, and A2B5 began to emerge after day seven (FIG.11B,C). This population, in which various neural progenitors (Alcock, J.et al., Cell Res 19(12):1324-1333 (2009)) appear to coexist, likelyrepresents the colony-forming cells (subset 2 in FIG. 11A). Notably,this population did not contain a significant number of cells expressingSSEA-1, a pluripotent stem cell marker (FIG. 11B). The heterogeneity ofthe reprogrammed cells (FIG. 7D) probably reflects parallel paths ofreprogramming, some of which result in a more directtransdifferentiation to mature neuronal fates—e.g. because matureneuronal cells coexist with transdifferentiated NPCs (FIG. 7D) andPSA-NCAM-expressing cells were generated as early as day seven in thenewly emerging population (FIG. 11C).

Following transdifferentiation, mitotically active neural colonies—whichhave typically been found to express Pax6 (Walther, C. et al.,Development 113(4):1435-1449 (1991))—were isolated by manual picking andsubcultured en bloc in conventional NPC medium containing FGF2 and EGFfor expansion of NPCs (FIG. 7B). Each small cluster could form a colonywithin several days after isolation; however, despite mild enzymaticpassaging and the avoidance of single cell dissociation, the NPCsappeared to lose their ability to form colonies within 3-5 passages. Atthis point, the NPCs were dissociated into single cells then cultured inN2 medium without any cytokines for further differentiation. After 1-2weeks of spontaneous differentiation, we observed NeuN andMap2-expressing mature neurons, GABAnergic neurons and GFAP-expressingastrocytes (FIG. 7E). These results collectively suggested that thePax6- and PLZF-expressing NPCs are functional NPCs that can bedifferentiated into mature neurons and glial cells. Compared to thecircuitous process of reprogramming to iPSCs and subsequentre-differentiation to NPCs, our induced transdifferentiationmethod—requiring only a single step that is complete within 13 days andshowing almost 100% newly generated colonies that are mostly comprisedof NPCs—is a highly efficient, direct and rapid process for generatingNPCs.

Importantly, the Nanog-GFP marker harboured by the secondary MEF cellswas never expressed during transdifferentiation. Indeed, its activationwas found to require at least nine days of dox treatment followed by aweek of subsequent culture (Wernig, M. et al. Nat Biotechnol26(8):916-924 (2008)). Thus, we concluded that no fully reprogrammediPSCs were generated within the time span of our experiment, and thatthe Pax6 and PLZF expressing cells were directly reprogrammed fromfibroblasts instead of re-differentiation from intermediate pluripotentcells. To better understand and characterize this transdifferentiationprocess, we performed additional analyses.

First, we sought to more definitively rule out the possibility that thegeneration of NPCs might first require the formation of transientpluripotent intermediates. To this end, we tested RepM-Neural treatmentdirectly on the iPSCs (NGFP1) which were used in blastocyst injectionsto make the secondary MEF cells, hypothesizing that we would get resultssimilar to MEFs if the generation of NPCs relied on the generation of asmall number of pluripotent cells early in the transdifferentiationprocess. However, we found that using iPSCs as a starting populationresulted in a highly complex mixture of neuroectodermal (Sox1- andPax6-positive), endodermal (Sox17-positive), and mesodermal (T-positive)cells (FIG. 8A,B). On the contrary, most colonies transdifferentiatedfrom MEFs were almost entirely comprised of cells expressing Sox1, theearliest neuroectodermal marker (Ying, Q. L. et al., Nat Biotechnol21(2):183-186 (2003)), and Pax6 (FIG. 8A,B). RT-PCR analysiscorroborated these results, as Sox1 and Pax6 expression was exclusive intransdifferentiation (FIG. 8D). As above, cells generated from iPSCsdisplayed a much more arbitrary profile of lineage-specific marker geneexpression (FIG. 8E, F). Although conventional neural differentiationdoes not rely on NPC-supporting cytokines, we could not do a comparisonin their absence because no cell growth of transdifferentiated cells wasever observed without these cytokines. These results show thattransdifferentiated NPCs arise directly from fibroblasts without anydependence on the generation of pluripotent intermediates.

Second, we analyzed neural marker expression over time to pinpoint theonset of neural specification during transdifferentiation. As early asday three, Pax6 expression was increased by 5.6-fold compared to D0MEFs. Sox1 expression started on day five and increased dramaticallythereafter (FIG. 8D). Critically, during iPSC differentiation, Sox1expression also begins on day five (FIG. 8F). This latter findingimplies that if transdifferentiation relied on the generation of apluripotent intermediate, this intermediate would have to arise at leastfive days before initial Sox1 expression. Since Sox1 expressioncommences on day five during transdifferentiation, this leaves no timefor the reprogramming of MEFs to pluripotency. Further, even minuteamounts of Nanog gene expression are not detected until much later inthe transdifferentiation process (FIG. 8C). In short, Sox1 NPCs cannotbe arising from putative pluripotent intermediates generated duringtransdifferentiation.

We also investigated the possible generation of epiblast stem cells(EpiSCs) during the transdifferentiation because FGF2 supportsself-renewal of EpiSCs, but could not detect any FGF5 expression(Greber, B. et al., Cell Stem Cell 6(3):215-226 (2010)) at any point inthe process (FIG. 8D). In addition, Sox17 and Brachyury (7), which arenot only lineage markers but are also highly expressed in EpiSCs (Tesar,P. J. et al., Nature 448(7150):196-199 (2007)), were not detected either(FIG. 8D). Collectively, our results strongly support the notion thattransdifferentiated NPCs are derived from non-pluripotent intermediatecells arising during the early phase of the process.

Third, we surmised that the fate choice between NPCs and iPSCs isdetermined by exposure to signalling specific to each cell type. To testthis hypothesis, we examined the effects of using LIF-containing medium(FIG. 9A). Strikingly, even brief exposure (as little as one day) to LIFclearly decreased the number of PLZF-expressing colonies on day nine(FIG. 9B). Conversely, flow cytometric analysis showed that LIF exposureincreases the number of SSEA1-expressing cells (FIG. 12B,C). Geneexpression analysis also showed down-regulation of Sox1 and Pax6 withconcomitant up-regulation of Nanog and Rex1 under the same conditions(FIG. 9C,D). The correlated expression between SSEA1 and Nanog as wellas Rex1 implies an overall increase in pluripotent character by extendedLIF treatment. Accordingly, we observed that small clusters of Nanog-GFPexpressing cells were generated when we used RepM-Pluri throughout(essentially ESC maintenance medium) instead of RepM-Neural.Interestingly, Sox17, T, and FGF5 expression on days 9, 11, and 13 wasalso increased by LIF exposure (FIG. 13B). This increase in expressionof genes specific to other lineages might result from the spontaneousre-differentiation of Nanog-GFP expressing cells generated by LIFexposure or perhaps from an induction of EpiSC-like cells. These resultsimply that intermediate cells may embark on the path towards becomingiPSCs or NPCs by responding to LIF or NPC-supporting medium,respectively. This cytokine-dependent cell-type specification duringdirect reprogramming has also been shown in other recent studies (Szabo,E. et al., Nature 468(7323):521-526 (2010); Han, D. W. et al., Nat CellBiol (2010)). Thus, it is clear that the choice between conversion toNPCs and complete reprogramming to iPSCs could be influenced at a veryearly point during the transdifferentiation process.

Finally, we did not observe any expression of pluripotency (Oct4),neural (Sox1, Pax6), or neuronal (Tuj1) markers in the starting cells orin the cells cultured in RepM-Neural media without dox induction,strongly suggesting an absence of contaminating neural cells in thestarting MEF population. Nonetheless, to rigorously rule out anycontribution from contaminating neural crest cells and mesenchymal stemcells that might exist in rather heterogeneous MEF populations (Weston,J. A. et al., Dev Dyn 229(1):118-130 (2004)), we reprogrammed adulttail-tip fibroblasts (TTFs) using the Dox-inducible STEMCCA system(Sommer, C. A. et al., Stem Cells 27(3):543-549 (2009)) following thesame method described above. We successfully transdifferentiated TTFsinto Pax6-expressing NPCs in 13 days (FIG. 9E) with an efficiency of0.07% (±0.006% s.e.m.) using just six days of induction—shorter than theeight days required for the generation of pluripotent cells from TTFwith this system (Sommer, C. A. et al., Stem Cells 27(3):543-549(2009)).

Because short-term LIF treatment seems to increase the pluripotentcharacter of the reprogrammed cells—although levels of Nanog and Rex1expression are still quite low compared to iPSCs (FIG. 9D)—a smallnumber of LIF-responsive cells might exist in our cultures. To strictlyeliminate the possibility of these cells, we inhibited JAK/Stat3signalling which is directly involved in pluripotent reprogramming(Yang, J. et al., Cell Stem Cell 7(3):319-328 (2010)) by treating smallmolecular inhibitor of JAK. Although this inhibition decreased thenumber of colonies by around 30% (FIG. 9F), all remaining colonieshomogeneously expressed Pax6 (FIG. 9E). These results imply that ourtransdifferentiation strictly takes independent path from thepluripotent reprogramming which can be blocked by JAK inhibitor)treatment.

In summary, we have successfully transdifferentiated fibroblasts intofunctional and proliferating NPCs. Such four iPSC-factors-basedtransdifferentiation can be guided by modulating transgene expressiontime and the culture environment (such as specific cytokines) and willprove useful for transdifferentiation to other lineages as well.

Discussion

We have shown a direct cell type switch from fibroblasts to functionalNPCs by transient expression of the four reprogramming factors, wherebythe process is clearly distinct from and does not depend on thegeneration of iPSCs. This transdifferentiation process is highlyspecific and efficient, reaching completion within 9-13 days. Our methodachieves an inter-lineage cell type change much like that of iN cells(Vierbuchen, T. et al., Nature 463(7284):1035-1041 (2010)), with onecritical advantage: the resulting cells are expandable progenitor cells.Another advantage of our method is the use of general reprogrammingfactors instead of lineage-specific transcription factors. Because thefour Yamanaka factors were chosen for pluripotent cell generation(Takahashi, K., et al., Cell 126(4):663-676 (2006)), they are generallyonly considered useful for the derivation of iPSCs. However, our resultssuggest a different paradigm in which various developmentally plasticintermediate cells may be generated in the process, and that iPSCs areperhaps only one of many possible outcomes of the four-factorreprogramming. The ultimate result may depend largely on extrinsicsignalling inputs (FIG. 10). Interestingly, in the newt, Sox2, Klf4, andc-Myc are temporarily up-regulated after lens removal or limb amputationto initiate the regeneration process (Maki, N. et al., Dev Dyn238(6):1613-1616 (2009)). This observation lends supports to ourhypothesis that the four conventional iPSC-factors not only inducereprogramming to iPSCs, but may also be capable of mediating direct fateswitching between differentiated cells. Indeed, we have also found thatfibroblasts can be transdifferentiated into spontaneously contractingcardiac cells by temporary expression of the same four reprogrammingfactors under different culture condition within 11 days (Efe et al., inpress). These results collectively imply a generallynon-specific/undirected reprogramming process induced by the fouriPSC-factors (i.e. not specifically directed toward the pluripotentstate as it has been regarded), and suggest a new strategy/paradigm tosignificantly expand and exploit iPSC-factor-based reprogramming.Different transgene expression time and culture conditions may allow atransient, plastic developmental state established early to effectivelyserve as a cellular platform for transdifferentiation toward variouslineages.

Although iN cells are functional neurons (Vierbuchen, T. et al., Nature463(7284):1035-1041 (2010)), they lack the potential to generate thediverse neuronal subtypes that can be derived from iPSCs. Furthermore,these post-mitotic neurons may not be very suitable to the study ofcertain neurological diseases, due not only to their non-proliferativestate (which severely limits their numbers), but also to their inabilityto recapitulate disease phenotypes occurring at the neural progenitorstage (Marchetto, M. C. et al., Hum Mol Genet (2010)). Currently, iPSCsderived from patients with late-onset neurological diseases (e.g. thosewith Parkinson's and amyotrophic lateral sclerosis) do not readilyrecapitulate disease phenotypes when re-differentiated (Soldner, F. etal., Cell 136(5):964-977 (2009) Dimos, J. T. et al., Science321(5893):1218-1221 (2008)), although some patients' iPSCs with geneticdefects show their symptoms (Lee, G. et al., Nature (2009); Ebert, A. D.et al., Nature 457(7227):277-280 (2009)). These findings imply thatcomplete reprogramming to a pluripotent state may reset certainepigenetic hallmarks of the disease state, thereby necessitatinglong-term aging under conditions of stress for its repeatedmanifestation. Considering this negative correlation between the degreeof reprogramming and the manifestation of a particular diseasephenotype, we think that the transdifferentiated NPCs—which are derivedwith limited reprogramming—may prove to be a superior disease modelsystem to iPSCs when studying such late-onset diseases.

Although we used secondary MEF cells, the transdifferentiation processmay at first glance seem inefficient compared to iPSC reprogrammingusing the same cells (Wernig, M. et al., Nat Biotechnol 26(8):916-924(2008)). However, considering the abbreviated induction period, theefficiency is reasonable, i.e. comparable to initial reprogramming asconfirmed by retrospective analyses (Smith, Z. D. et al., Nat Biotechnol28(5): 521-526 (2010)).

We observed no significant up-regulation of EpiSC markers such as Sox17,Brachyury (Tesar, P. J. et al., Nature 448(7150):196-199 (2007)) andFGF5 (Greber, B. et al., Cell Stem Cell 6(3):215-226 (2010)) during ourdirect reprogramming. Nonetheless, a recent study by the Schöler groupshowed direct reprogramming to EpiSCs can be achieved in 3-5 weeks (Hanet al., Nat Cell Biol, 13(1): 66-71 (2011)) which is substantiallyslower than our method. Thus, a temporary emergence of EpiSCs during ourdirect reprogramming—which can be achieved within 12 days—is highlyunlikely. Interestingly, five days of induction followed by long-termLIF treatment caused increased expression of Sox17 and FGF5 withextremely low levels of Brachyury and Nanog (FIG. 13). These cells maypartially resemble EpiSCs; however, the absence of LIF in ourtransdifferentiation media makes the generation of such cells nearlyimpossible. To achieve fully reprogrammed EpiSCs or iPSCs status, a muchlonger induction of reprogramming factors may be required.

In a recent exciting development, lentiviral expression of OCT4 alonewas shown to mediate direct reprogramming of human cells to bloodprogenitors (Szabo, E. et al., Nature 468(7323):521-526 (2010)).Although OCT4 expression is down-regulated after long-termdifferentiation, long-term induction is required and there is always arisk of lentiviral reactivation of OCT4 expression, which may inducedysplastic lesions (Hochedlinger, K. et al., Cell 121(3):465-477(2005)). Our study shows that temporary expression of four reprogrammingfactors is sufficient to induce lineage-specific transdifferentiation.When our transdifferentiation process can be replicated using non-viraland temporary expression methods such as transient transfection (Okita,K. et al., Science 322(5903):949-953 (2008); Jia, F. et al., Nat Methods7(3):197-199 (2010)), protein transduction (Zhou, H. et al., Cell StemCell 4(5):381-384 (2009); Kim, D. et al., Cell Stem Cell 4(6):472-476(2009)), mRNA transfection (Warren, L. et al., Cell Stem Cell7(5):618-630 (2010)) or small molecules (Zhu, S. et al., Cell Stem Cell7(6):651-655 (2010)), it will form the basis of safe and convenienttransdifferentiation across a broad lineage spectrum.

Materials and Methods

Direct reprogramming and differentiation: NGFP1 Dox-inducible iPS cells(Hanna J. et al., Nature (2009); Hanna, J. et al., Cell 133(2):250-264(2008)) (Stemgent) were injected into C57BL/6 blastocysts and implantedinto surrogate mice (CD1, Harlan). Chimeric embryos were isolated atE12.5-13.5. Heads, developing organs and spinal cords were carefullyremoved from the embryos and MEFs were prepared and selected aspreviously described (Hanna J. et al., Nature (2009); Wernig, M. et al.,Nat Biotechnol 26(8):916-924 (2008); Hanna, J. et al., Cell133(2):250-264 (2008)). For reprogramming, secondary MEFs (p3-4) wereplated on Geltrex-coated culture dishes at 1.5-2×10⁴ cells/cm² in MEFmedium (Dullbecco's modified Eagle's medium supplemented with 10% fetalbovine serum, 0.1 mM non-essential amino acids, and 2 mM Glutamax).Doxycycline (Sigma-Aldrich, 2-8 μg/ml) treatment was initiated the nextmorning (Day 0) and continued until the designated day(s). Cells werecultured in MEF medium for an additional day and changed toreprogramming initiation medium (RepM-Ini; knock-out DMEM supplementedwith 10% knock-out serum replacer, 5% FBS, 0.1 mM NEAA, 2 mM Glutamax,and 0.055 mM β-mercaptoethanol) for three days. For neuralstem/progenitor reprogramming, the medium was changed into neuralreprogramming medium (RepM-Neural; Advanced DMEM/F12 and Neurobasalmedium were mixed by 1:1 and supplemented with 0.05% bovine serumalbumin, 1×N2, 1× B27, 2 mM Glutamax, 0.11 mM β-mercaptoethanol, 20ng/ml FGF2, 2 ng/ml FGF4, and 20 ng/ml EGF). RepM-Pluri is the same asRepM-Ini, with the addition of 1000 units/ml LIF (Millipore).

For TTF reprogramming, Lentivirus supernatants were produced andharvested as previously described (Takahashi K. et al. Cell131(5):861-872 (2007)). Briefly, 293T cells (Invitrogen) were plated at5×10⁶ cells per 100 mm dish and incubated overnight in a 37° C.incubator. Cells were transfected with 4 μg pHAGE2-TetOminiCMV-STEMCCA(29) or FUW-M2rtTA (Addgene) along with packaging mix (1 μg psPAX2 and 3μg pMD2.G) (Addgene) and FuGENE 6 Transfection Reagent (Roche),according to the manufacturer's instructions. The cells were thencultured in a 32° C. degree incubator, after which supernatants werecollected at 48 and 72 hours (post-transfection) and filtered through a0.45 μm filter (Millipore) before use. TTFs were prepared as previouslydescribed (Takahashi, K., et al., Cell 126(4):663-676 (2006)). Briefly,tails (only the final third) were cut into small pieces (less than 0.5cm) and grown on gelatine-coated plates in MEF medium. Cells thatmigrated out of the tissue were transferred into new dish and used afterone more passage (p3). After two rounds of transduction with STEMCCA andrtTA expressing virus, cells were plated on Geltrex-coated dishes at2×10⁴ cells/cm² and reprogrammed as described above. JAK inhibitor 1(Calbiochem) was added from day 4 onward at 0.5 μM during TTFtransdifferentiation.

For neuronal differentiation, completely dissociated cells were platedon Geltrex-coated plates in N2 medium (Elkabetz, Y. et al., Genes Dev22(2):152-165 (2008)). For differentiation, iPSCs cultured in RepM-Pluriwere dissociated into single cells and plated on Geltrex-coated plates.After overnight culture, the medium was replaced with RepM-Neural. Allmedia were replenished at least once every two days. All reagents werepurchased from Invitrogen if not specified, and all cytokines are fromR&D systems.

Quantitative RT-PCR: Total RNA was extracted from samples at thedesignated time points using the RNeasy Plus Mini Kit with QiaShredder(Qiagen). One microgram of total RNA per sample was reverse-transcribedusing the iScript cDNA synthesis kit (Bio-Rad) and the cDNA was dilutedwith 130 ul of water. 1/50 of the diluted cDNA was used for quantitativePCR with iQ SYBR Green Supermix on the CFX96 system (Bio-Rad). All qPCRreactions were done in duplicate or triplicate, and the expression datawere normalized to both Gapdh and β-actin expression using CFX managersoftware (Bio-Rad). All primer sequences are listed in previouspublications (Hanna J. et al., Nature (2009); Wernig, M. et al., NatBiotechnol 26(8):916-924 (2008); Greber, B. et al., Cell Stem Cell6(3):215-226 (2010)).

Immunocytochemical analysis: Samples were washed once with D-PBS(Invitrogen, without Ca²⁺ and Mg²⁺) and were fixed with a 4%pure-formaldehyde solution (Electron Microscopy Sciences) with 0.15%picric acid (Sigma-Aldrich) in D-PBS for 20 min, followed by threewashes with D-PBS. Blocking and permeabilization were done with a 3% BSA(Jackson ImmunoResearch) and 0.3% Triton X-100 (Sigma-Aldirch) solutionin D-PBS for 1 hr at room temperature. All primary antibodies werediluted in 1% BSA and incubated overnight at 4° C. After 1 hr of washing(including several buffer changes) with 0.1% BSA in D-PBS, samples wereincubated with Alexa-555- or Alexa-488-conjugated secondary antibodies(Invitrogen) for 1 hr at room temperature. All images were taken using aZeiss AX10 microscope equipped with an Axiocam HRm camera, and processedwith Axiovision software (Zeiss). Primary antibodies used are Pax6(Covance, 1:500, rb), Tuj1 (Covance, 1:5000, m or rb), Dcx (SantaCruz,1:200, gt), TH (Sigma, 1:500, m), ZO-1 (Invitrogen, 1:500, rb), PLZF(EMD chemical, 1:100, m), GFAP (Dako, 1:1000, rb), GABA (Sigma, 1:3000,rb), NeuN (Millipore, 1:50, m), Map2 (Abcam, 1:5000, chk), Sox1(SantaCruz, 1:300, gt), Sox17 (R&D, 1:1000, gt), and T (SantaCruz,1:300, gt).

Flow cytometric analysis: Cells cultured until the designated timepoints were washed with D-PBS and dissociated with Accutase (InnovativeCell Tech). After harvesting, the cells were washed twice with ice-coldFACS buffer (HBSS supplemented with 10 mM HEPES, 2% FBS, and 0.1% SodiumAzide [Sigma-Aldrich]). Undissociated cells were removed by passingsuspensions twice through a strainer (BD). Cells were incubatedindividually with PE-conjugated SSEA1 (BD) and APC-conjugated PSA-NCAM,Prominin-1 and A2B5 (Miltenyi Biotec) antibodies for 30 min at 4° C.(all concentrations as suggested by the manufacturer). Appropriateisotype control antibodies were also used separately. After incubation,cells were washed twice with five volumes of FACS buffer, fixed, andresuspended in 4% pure-formaldehyde solution (Electron MicroscopySciences) in PBS. More than 20,000 cells were analyzed using FACSCaliburand CellQuest software (BD). For further analysis, FlowJo software (TreeStar) was used.

Example 4 Direct Reprogramming of Human Fibroblasts to Endodermal CellsExperimental Procedures

Direct reprogramming of human fibroblasts: Human fibroblasts (CRL-2097)were cultured in a 100 mm tissue culture dish and transduced withfreshly produced virus supernatants, as previously described. Then10,000 OCT4 transduced cells were seeded on the Matrigel coated plateand cultured in reprogramming medium and treated with 304 CHIR99021(Stemgent) for 1 week, followed by treatment with 304 CHIR99021, 0.1 mMNaB and 100 ng/ml Activin A for another 3-4 weeks. The CXCR4-positivecolonies were picked up for expansion in Expansion medium and passagedat the ratio of 1:3 each time by

Accutase. Reprogramming medium: Advanced DMEM/F12, 10% Knockout serumreplacement, 1% Glutamax, 1% Non-essential amino acids, 1%penicillin/streptomycin, 0.1 mM β-mercaptoethanol. Expansion medium:Advanced RPMI, 1% Glutamax, 1% Non-essential amino acids, 0.5×N2, 0.5×B27, 1% penicillin/streptomycin, 0.1 mM β-mercaptoethanol, 25 ng/mlWnt3a, 50 ng/ml EGF and 100 ng/ml Activin A (Stemgent). All cell cultureproducts were from Invitrogen/Gibco BRL except where mentioned.

Production of pancreatic β-like cells: The protocol for furtherinduction of pancreatic β-like cells was modified from previouslypublished literatures. Briefly, iDEs were cultured in medium I andtreated with 50 ng/ml FGF7, 2 μM Retinoic acid (RA), 0.1 mM GDC-0449 (aHedgehog pathway inhibitor), 0.1 mM LDN-193189 (a BMP inhibitor) and 0.5μM A83-01 (a TGFβ/ALK5 receptor inhibitor) for 5 d; then changed tomedium II adding 50 ng/ml EGF and 104 DAPT for 3 d; finally, these cellswere cultured in medium III with 10 ng/ml bFGF, 50 ng/ml Extendin-4 and10 mM Nicotinamide for another 5-7 d. Medium I: IMDM/F12, 1% Glutamax,0.5% BSA, 0.5×ITS, 0.5× B27, 1% penicillin/streptomycin; Medium II:DMEM, 1% Glutamax, 1× B27, 1% penicillin/streptomycin; Medium III:DMEM/F12, 1% Glutamax, 1× B27, 1% penicillin/streptomycin.

Results

Direct reprogramming of human fibroblasts to definitive endoderm (iDE):Induced definitive endoderm (iDE) could be generated from human primaryfibroblasts by OCT4 and a unique endodermal inducing condition (304CHIR99021 (a GSK-3 inhibitor), 0.1 mM NaB (an HDAC inhibitor) and 100ng/ml Activin A (a TGFβ/Activin/Nodal family member)). These iDEs wereboth SOX17 and FOXA2 positive, a typical characterization for definitiveendoderm (FIG. 14A). Bisulfate sequencing analysis revealed that theSOX17 and FOXA2 promoters of iDEs were largely demethylated, providingfurther evidence for reactivation of the endodermal transcriptionprogram in iDEs (FIG. 14B). Additionally, these iDEs expressed specificgenes of definitive endoderm, including SOX17, FOXA2 and CER, but didnot express pluripotent genes, such as OCT4 and NANOG (FIG. 14C). Inconclusion, these iDEs represent typical definitive endodermal cells.

Further generation of pancreatic β-like cells: To further investigatethe development potentials of these iDEs, we applied a modified protocolfor inducing these iDEs to pancreatic-lineage cells (FIG. 15A). Afterabout 2 weeks of step-wise induction and maturation, pancreatic β-likecells were finally generated from these iDEs (FIG. 15B). Thesepancreatic β-like cells expressed typical lineage-specific markers, suchas PDX1, NKX6.1, C-PEPTIDE and INSULIN. These INSULIN-positive cellswere also C-PEPTIDE positive, excluding insulin uptake from theculturing media. Real-time PCR analysis showed that these pancreaticβ-like cells also expressed islet specific genes, including PDX1,NKX6.1, MAFA, GLUT2, GLUCOKINASE and INSULIN (FIG. 15C). Furthermore,human C-peptide was released from these pancreatic β-like cells onglucose stimulation in vitro, indicating that these pancreatic β-likecells are functional (FIG. 15D). In conclusion, these iDEs could befurther induced to insulin producing and glucose responsive pancreaticβ-like cells.

Example 5 Direct Reprogramming of Mouse Fibroblasts to Endodermal CellsMethods of Differentiation

For the preparation of secondary mouse embryonic fibroblasts, NGFP1Dox-inducible iPS cells (Hanna et al., Nature, 26(8):916-924 (2009);Hanna et al., Cell, 133(2):250-264 (2008)) were injected into C57BL/6blastocysts and implanted into surrogate mice (CD1, Harlan). Chimericembryos were isolated at E12.5-13.5. Heads and developing organs werecarefully removed from the embryos and mouse embryonic fibroblasts wereprepared and selected as previously described (Hanna et al., Nature,26(8):916-924 (2009); Hanna et al., Cell, 133(2):250-264 (2008); Werniget al., Nat. Biotechnol., 26:916-924 (2008)).

Secondary MEFs (p2-3) were plated on matrigel coated culture dishes at1×10⁴ cells/cm2 in MEF media. The medium was changed to Medium I withDoxycycline (4 m/mL) on the next day (Day 0). On Day 5, medium waschanged to Medium I with Activin A (10 ng/ml) and JAK inhibitor (0.5μM). On Day 9, medium was changed to Medium II with RA(2 μM), A83-01(0.5μM), BMP4(10 ng/ml) and bFGF(10 ng/ml). On Day 12, medium was changed toMedium III (see FIG. 16). Cells were feed every 2 days throughout thewhole process. MEF media: DMEM, 2 mM Glutamax, 10% FBS, 1×penicillin/streptomycin. Medium I: Knockout DMEM, 2 mM Glutamax, 10%KSR, 5% FBS, 0.1 mM NEAA, 0.11 mM β-mercaptoethanol, 1×penicillin/streptomycin. Medium II: DMEM, 2 mM Glutamax, 1× B27 mediasupplement, and 1× penicillin/streptomycin. Medium III: DMEM/F12, 10%FBS, 20 nM progesterone, 100 μM putrescine, 1 mg/mL laminin, 10 mMnicotinamide, 1×ITS premix containing insulin, transferrin, and selenicacid, 1× B27 media supplement, and 1× penicillin/streptomycin.

Results

On Day 25, cells were fixed for C-peptide immunostaining (FIG. 16).About 50% of the cells were C-peptide positive.

Example 6 Cardiomyogenesis in Pluripotent Stem Cells Methods ofDifferentiation

Pluripotent ESCs represent a potentially unlimited source of functionalcardiomyocytes since they can be proliferated in the pluripotent stateindefinitely and can be differentiated into beating cardiomyocytes.Realization of this potential is contingent upon the development ofconsistent and efficient methods of differentiation. The most commonmethod of inducing cardiomyogenesis of murine ES cells involvessuspension culture (in the presence of serum and absence of supplementedleukemia inhibitory factor/LIF) to form aggregates (EBs), whichdifferentiate into various cell types, including hematopoietic,endothelial, neuronal and cardiac muscle cells [3]. Isolation of thedesired cell type can be achieved through EB dissociation and FACS,followed by replating. When used in combination with specific treatmentsof growth factors, this has been an effective, though cumbersome, methodof generating cardiac cell types and has been particularly useful in theidentification of cardiac precursor cells. While the differentiationprotocols described above have great utility, they are still not optimalfor the study of factors influencing cardiac specification. Inparticular, media components such as fetal bovine serum (FBS) andKnockout Serum Replacement (KO-SR) contain undefined compositionsincluding growth factors; for example KO-SR has been shown to have bonemorphogenetic protein (BMP) activity. The use of undefined media canobscure and complicate the analysis of factors affecting cardiomyocytedifferentiation. Additionally, the 3-dimensional nature of the EB modelof cardiomyocyte differentiation makes it difficult to obtainhomogeneous populations of cells due to the presence of diffusiongradients within embryoid bodies and three-dimensional cell-cellinteractions. As EB differentiation is similar to that of the developingembryo, it inherently involves the generation of heterogeneouspopulations of cells. In contrast, monolayer differentiation provides agreater ability to provide a specific and uniform local concentration ofgrowth factors.

An alternate strategy to EB differentiation that has proven successfulin the human system is the coculture of ESCs with a cell line that candirect cell fate. In particular, by culturing the hESC lines hES2 andhES3 with a mouse endoderm-like cell line (END2), an average of 25%cardiomyocytes can be derived. [9] The authors speculate that unknownfactors secreted by the endodermal cells, may be directly stimulatingthe differentiation of cardiomyocytes. Unfortunately, the coculturemethod is not a defined system, and as such, it is difficult to pinpointthe underlying mechanisms of its success.

A method of differentiation which has proven successful indifferentiation of neural and endoderm derivatives has been monolayerdifferentiation in serum-free media. As discussed, serum-free mediaafford precise and repeatable determinations of the effectiveness ofgiven signaling mechanisms in cell fate specification. As such,considerable progress has been made using these systems ofdifferentiation to understand the mechanisms governing ectoderm andendoderm derivative specification. To date, similar success has yet tobe achieved with the differentiation of cardiomyocytes, and as such,there are several outstanding questions regarding cardiac fatespecification.

Signaling Mechanisms Involved in Cardiomyogenesis Wnt Signaling

While the importance of the canonical Wnt pathway in cardiomyogenesishas long been appreciated, the modality in which it promotescardiovascular development is unclear as both activators and inhibitorsof Wnt signaling have been reported to have positive effects.Experiments with chick embryos demonstrated that the Wnt antagonistsDickkopf-1 and Crescent, released by the adjacent anterior endoderm,stimulate the differentiation of cardiac mesoderm. Similarly, in frogembryos, Dickkopf-1 and Crescent diffuse from Spemann's organizer andstimulate cardiac differentiation in the adjacent mesoderm. In mouseembryos as well, conditional inactivation of β-catenin in the definitiveendoderm resulted in ectopic heart formation [10]. On the other hand,Wnt signaling is known to be essential to all aspects ofcardiomyogenesis in drosophila. Studies concurrent with our own, havesuggested a biphasic role of Wnt signaling in cardiac development in themouse.

BMP Signaling

Much like Wnt signaling, the importance of BMP signaling in cardiacdevelopment is appreciated though the specific mechanisms are unclear.Initial studies in amphibian and chick embryos demonstrated that BMPsignaling mediates cardiac specification by the adjacent endoderm [15].BMP signaling was subsequently shown to be essential in vertebrate heartformation, too [16]. Conversely, treatment with the BMP inhibitor Nogginresulted in a marked increase in cardiomyogenesis in an EB model ofmouse ES cell differentiation [17]. This study aims to identify amonolayer serum-free condition for the differentiation of cardiomyocytesthat can be used to clarify the specific factors and signaling requiredfor stimulation of this transition.

TGF-Beta/Activin/Nodal

In a series of studies, Keller used an ES cell line marked with GFPtargeted to the mesendodermal specific gene brachyury to quantifymesoderm and endoderm induction and to isolate and characterize thesepopulations. In short, it was found that in conjunction with Wnttreatment, Activin could indeed induce the formation of primitive streaklike cells (Bry+). Furthermore, using a doubly marked GFP-Bry CD4-Foxa2ES cell line, they were able to assess whether the primitive streakcells formed were anterior (high Foxa2) or posterior (low Foxa2). It wasdetermined that higher concentrations of Activin A stimulated theformation of anterior primitive streak cells; cardiac fate was isolatedto cells in this fraction.

Small Molecules Affecting Cardiac Differentiation

Using mESCs stably transfected with the cardiac muscle specific α-myosinheavy chain (αMHC) promoter-driven enhanced green fluorescence protein(EGFP) as a reporter, Takahashi et al. screened 880 known drugs inmonolayer culture and found that ascorbic acid (vitamin C) cansignificantly enhance spontaneous cardiac differentiation of mESCs.Interestingly, other antioxidants such as N-acetylcysteine or vitamin Edo not have a similar effect, thus suggesting that the cardiomyogenesisinducing activity of ascorbic acid may be independent of itsantioxidative property.

Concurrent with this work, the Schultz lab screened large combinatorialchemical libraries using P19 cells that were stably transfected with thecardiac muscle specific atrial natriuretic factor (ANF) promoter-drivenluciferase reporter, and found a series of diaminopyrimidine compounds,named cardiogenol A-D, that can efficiently and selectively induce P19and mESCs to differentiate into cardiomyocytes. The differentiated cellsexpressed multiple cardiac muscle markers, including GATA-4, Nkx2.5,MEF2, and myosin heavy chain (MHC), and formed large areas ofspontaneously beating patches.

Using P19 embryonic carcinoma cells with a stably integratedNkx2.5-firefly luciferase BAC, Olson et al. screened the 147,000compounds University of Texas Southwestern chemical library to findactivators of cardiac fate. The researchers initially found NaB to be anactivator of Nkx2.5-luc expression and thus used this as a positivecontrol in their screen. Using a chemoinformatic approach, theresearchers identified a particular family of sulfonyl-hydrazone (Shz)small molecules as being the most promising. The molecules were shownnot to be HDAC inhibitors, did not activate the CMV promoter in P 19CL6cells, and could not direct neuronal differentiation in stem cells.

In an in vivo small molecule screen aimed at identifying chemicals thatmight modulate dorsoventral patterning, the group recently identifieddorsomorphin(6-[4-(2-Piperidin-1-yl-ethoxy)phenyl]-3-pyridin-4-yl-pyrazolo[1,5-a]pyrimidine),a molecule which can selectively inhibit the BMP type I receptor. Sincethe growth factor Noggin, an inhibitor of BMP signaling, had previouslybeen shown to promote cardiac differentiation in an EB model of mousedevelopment, the authors tested dorsomorphin for cardiac inducingeffects. The authors report that treatment of ES cells with dorsomorphinfor only the first 24 hours of differentiation induced robust cardiacdifferentiation, while Noggin treatment had been reported to beeffective when treated for a 5 day span including 3 days beforedifferentiation. The authors postulate that dorsomorphin induces cardiacdifferentiation by restricting differentiation of other cell typesincluding the endothelial, smooth muscle, and hematopoietic lineages.

Small Molecule Screen for Regulators of Cardiomyogenesis

Generation and characterization of an Nkx2.5-GFP ESC line: One of thechallenges of assessing differentiation efficacy and efficiency is themethod of determining which cell types are generated. Common analyseswhich identify cell type include RT-PCR, western-blot, immunostainingand functional characterization (eg. beating cells). With the exceptionof some functional characterizations, all of these methods involve thetermination of the experiment as the cells must be harvested or fixed.An alternative strategy is to mark the cells with a fluorescent protein(GFP) under the control of a promoter specific for the cell type ofinterest. In choosing a marker for a cell type, it is usually best tochoose the earliest expressed gene that is also specific for the celltype, often a transcription factor gene as these generally precede theexpression of structural genes. In addition to being specific, themarker should also be ubiquitously expressed in the specific lineage ofinterest. Numerous transcription factors are expressed in the developingheart where they direct and control cardiac specific gene expression andregulate cell fate. Perhaps the most quintessential cardiac specifictranscription factor is Nkx2.5. Nkx2.5 is expressed throughout heartdevelopment as well as in cardiomyocytes. Additionally, the Nkx2.5 genehas ubiquitous expression in the heart tube, unlike some other cardiaclinked transcription factors (Tbx5) that can be expressed sporadicallyin the anterior-posterior axis of the heart tube.

Construction of the Nkx2.5-GFP ES cell: A pCSX-EGFP-PP-DT targetingplasmid containing an Nkx2.5 EGFP vector was obtained from the Morisakigroup in Osaka, Japan as seen in FIG. 17.

Of all the clones selected as having inserted the knock-in vector, 10cell lines with the correct GFP insertion into the Nkx2.5 locus wereexpanded for testing. Hanging drop embryoid bodies were generated foreach cell line and monitored daily for GFP expression. Unfortunately,GFP expression was not clearly visible in any of the clones, as is shownin FIG. 18 (nuclei stained blue with DAPI in a contracting region thatis negative for Nkx2.5-GFP expression).

Cells were differentiated further into beating colonies which alsoshowed levels of GFP expression that were marginally visible under thefluorescent microscope. As such, we determined that these cells would beinappropriate for the monitoring of cardiac differentiation inhigh-throughput screening setting. Further research with a collaborator(Burcin, Novartis) indicated that despite its crucial role in cardiacdevelopment Nkx2.5 transcript levels only increase 10 fold over therebasal levels (FIG. 19) while differentiating whereas the structural geneMLC-2v is more than 1000 fold upregulated cardiomyocytes.

Having determined that Nkx2.5 would not be an appropriate marker forcardiac differentiation, we sought to identify other markers that mightbe more suitable. Our collaborator at Novartis, Mark Burcin, hadrecently developed an MLC-2v-GFP ES cell line, that his lab had yet tocharacterize. Similar to the validation of the Nkx2.5 lines, wegenerated hanging drop EB's with the MLC-2v-GFP cells in order tospontaneously differentiate cardiomyocytes. Unlike transcriptionfactors, a structural gene such as myosin should be very highlyupregulated above basal levels. Additionally, as MLC-2v tends to markventricular cells (those most affected in a myocardially infractedheart) we reasoned that this would be an excellent marker fordifferentiation. Upon examination of beating regions of cells under thefluorescent microscope, GFP expression was observed in the contractingcells, as well as in some cells surrounding the contracting cells. Shownin FIG. 20 is a representative image of MLC-2v—GFP expression of EBdifferentiated cardiomyocytes with blue nuclei stained with DAPI). Whilewe hoped for a more specific pattern of expression, we felt these cellswould be sufficient to proceed with a small molecule high-throughputscreen.

To set up a screen for molecules capable of inducing cardiomyogenesis inES cells, we first sought to identify a permissive condition. Ideally, apermissive condition is one in which a low percentage of cellsdifferentiate into cardiomyocytes in the absence of a positive control.Too high of a percentage would mean the signal of a ‘hit’ molecule wouldnot be significantly higher than the background signal, and too low of apercentage could mean that the condition would not allow cardiacdifferentiation, and potential hits would thus be missed. We decided touse 384-well plates, as these had sufficient area for cell survival andcell homogeneity. As serum is capable of inducing mesodermaldifferentiation, we opted to use a media similar to ES cell mediawithout the self-renewal factor LIF. Various plating densities weretested and it was determined that 5,000 cells per well was sufficientlydense, without being overly dense causing cells to overgrow and die.Media was replaced every 3 days, and cells were observed under thefluorescent microscope to identify the ideal time for GFP observation.By day 15, we observed approximately 1% of cells expressed in GFP,though no beating regions were observed. Shown in FIG. 21 is a smallpatch of GFP positive cells differentiated in chemically defined mediumon Matrigel in a serum-free defined medium.

For the screen, 5,000 cells were plated per well of a gelatin coated,black 384-well plate, in 80 uL of ES media. The following day, cellswere washed with PBS, and the differentiation medium was added. We choseto use a library of 50,000 kinase directed compounds that had provedfruitful in previous similar studies. Using a pintool machine (FIG. 22),500 nl of compound was added to each well, and media was refreshed every3 days thereafter.

Approximately 280 compounds displayed a significantly higher amount ofsignal in the GFP channel of the microscope at 15 days. Shown in FIG. 23is a representative hit compound showing extensive signal in the GFPchannel of the fluorescent microscope.

False positives and false negatives can be a significant problem insmall molecule screens. False positives may include fluorescentmolecules that mimic the GFP signal, random differentiation in a givenwell, or interaction of a small molecule with a random component of themedium. False negatives may include compounds that had their effectmasked by a component of the medium. In order to screen out falsepositives, the initial 280 compounds were arrayed into a new 384-wellplate, and the screen was repeated using these. Minutes after addingcompounds, the cells were observed under the fluorescent microscope toassess possible compounds fluorescence. The 15 strongest fluorescingcompounds of the original 280 were inducing fluorescence in the cellsonly 5 minutes after addition, indicating a false positive due tocompound fluorescence. Of the rest, only 10 of these initial compoundsrepeated in the first follow-up, and unfortunately none of the 10repeated in a second follow-up. We theorized that the lack of repeatablehits in this screen was due to a combination of false positives andfalse negatives, with the underlying problem being undefined and randommedia components (serum) that mask real hits, and stimulate false hits.

A Serum-Free, Monolayer Condition for Cardiomyocytes:

The most common method of inducing cardiomyogenesis of murine ES cellsinvolves suspension culture (in the presence of serum and absence ofsupplemented leukemia inhibitory factor/LIF) to form aggregates (EBs),which differentiate into various cell types, including hematopoietic,endothelial, neuronal and cardiac muscle cells. Isolation of the desiredcell type can be achieved through EB dissociation and FACS, followed byreplating. When used in combination with specific treatments of growthfactors, this has proven to be an effective, though cumbersome, methodof generating cardiac cell types and has been particularly useful in theidentification of cardiac precursor cells. While the differentiationprotocols described above have great utility, they are still not optimalfor the study of factors influencing cardiac specification. Inparticular, media components such as fetal bovine serum (FBS) andKnockout Serum Replacement (KO-SR) contain undefined compositionsincluding growth factors. For example KO-SR has been shown to have bonemorphogenetic protein (BMP) activity. The use of undefined media canobscure and complicate the analysis of factors affecting cardiomyocytedifferentiation. Additionally, the 3-dimensional nature of the EB modelof cardiomyocyte differentiation makes it difficult to obtainhomogeneous populations of cells due to the presence of diffusiongradients within embryoid bodies and three-dimensional cell-cellinteractions. As EB differentiation is similar to that of the developingembryo, it necessarily involves the generation of heterogeneouspopulations of cells. In contrast, monolayer differentiation provides agreater ability to provide a specific and uniform local concentration ofgrowth factors.

Assay Development:

While cellular differentiation is a fluid process, its study can besimplified by defining stages of differentiation. In order to identifyconditions conducive to cardiomyocyte differentiation, we employed amatrix strategy in which cells were differentiated for 10 daysencompassing 4 treatment windows (followed by a period of maturation inthe final procedure). The stages identified for the current study weremesendoderm, mesoderm, cardiac precursor, early cardiomyocyte, andmature cardiomyocyte. Shown in FIG. 24 is a schematic representing thetransition from ES cells to cardiomyocytes, including markers for eachcell type defined and the treatments used to stimulate each transition.

We began by identifying a minimal serum-free condition in whichapproximately 1% of cells would express αMHC (alpha-myosin heavy chain)after 10 days of culture. Combinations of medium components and platecoatings were tested in parallel, with the most permissive andconsistent condition being a modified chemically defined N2/B27 mediumon Matrigel coated plates (see Methods). Prior to differentiation, EScells were maintained in a pluripotent state on MEFs and with LIF. Atday −1, the cells were trypsinized and replated on Matrigel coatedplates in LIF supplemented medium, allowing for better cell survival andmore robust initial colony formation. In one day, a sufficient colonysize is achieved allowing survival in serum-free medium (FIG. 25).

From a literature search, we identified growth factors, cytokines andsmall molecules reported to influence cardiac differentiation, such asactivators and inhibitors of BMP, TGF-β/Nodal, Hedgehog, Wnt, and Notchpathways and tested each for their ability to induce cardiomyogenesis ateach time window. For example, to test Dkk-1 in the second window, cellswere in basal medium for days 0-2, treated with Dkk-1 for days 2-5, thencultured in basal medium until day 10 before immunofluorescence scoring.Positive regulators of cardiac specification at each stage weretherefore identified by their ability to induce alpha-myosin heavy chain(αMHC) expression at day 10, as assessed by immunostaining

Results BMP and Wnt Stimulate Early Cardiac Differentiation

Some factors had no significant effect (e.g., hedgehog), few hadnegative effects (e.g., Activin A), while two factors had strikinglypositive effects in directing cardiac specification. The potent GSK-3inhibitor, 6-bromoindirubin-3′-oxime (BIO) (FIG. 26), was found toincrease the percentage of αMHC staining when administered in the firstwindow (day 0-2), and to a lesser extent during the second window (day2-5), with an optimal dosage of 4 uM. Shown in FIG. 26 is arepresentative image of BIO induced cardiac cells stained for alpha-MHCand GATA-4.

BMP-4, a member of the TGF-β superfamily, had similar effects on thepercentage of αMHC positive cells when administered during the firstand/or second treatment windows with an optimal concentration of 20ng/ml. Shown in FIG. 28 is a representative image of BMP-4 inducedcardiac cells stained for alpha-MHC and GATA-4.

Treatment with either BMP-4 or BIO in the first window resulted inapproximately 20% of cells staining for αMHC. When the optimaltreatments from these first two windows are combined into a singledifferentiation scheme, the cells develop and differentiate intocolonies where approximately 35% of cells stain positive for MHC at day10.

Inhibition of Wnt by Dkk-1 Stimulates Late Cardiac Differentiation

Having identified positive effectors of cardiomyogenesis in the firsttwo windows of differentiation, we sought to reexamine our group offactors in the later stages of differentiation. In this iteration, cellswere treated with a combination of BMP-4 and BIO for 5 days before beingtreated in the third window (day 5-7) or fourth window (day 7-10). Inthis case, we found that treatment with the Wnt inhibitor Dkk-1 furtherincreased the efficiency of cardiac differentiation by 20%. Furthermore,when these colonies of cells were replated onto gelatin coated platesand maintained in a basal medium, nearly 50% developed a beatingphenotype after 6 days of culture.

Time Course Analysis of Differentiation

In order to arrive at a clearer understanding of this process, wecarried out immunofluorescence and RT-PCR studies of the differentiatingcells. At day −1, the cells were replated onto Matrigel in LIFcontaining medium, and by day 0 the cells continued to express thepluripotency marker Oct-4 (FIG. 29). At this point, the self-renewalcues were removed and mesoderm specification began through simultaneousBMP and Wnt signaling. At this stage, cells began to resemble theprimitive streak of the developing embryo, expressing markers such asBrachyury T and Gata-4, with Gata-4 being expressed as early as day 2(FIG. 29) and Brachyury T being expressed as early as day 3 (FIG. 18).

Listed in Table 1 are the specific primers and conditions used for theRT-PCR studies.

TABLE 1  RT-PCR Primers and Conditions Melting Gene Sense AntisenseCycles T (° C.) Oct3/4 GGCGTTCTCTTTGGAAAG CTCGAACCACATCCTTCT 25 58GTGTTC CT BryT CAT GTA CTC TTT CTT GGT CTC GGG AAA GCA 33 58 GCT GGGTG GC GATA4 TTC CTT GTC CTC ATC GAC AA TGT TAA CGG 32 58 ACC CAC AGAGTT GTG GAG G Flk1 GGTTCTCTGTCAAGTGGC AGCACACAGGCAGAAAC 32 58 GGTAAACAGTAGA Isl-1 TGT CAG GAG ACT TGC GCC AAA CGT TTA TTA 32 58 CAC TTTGTG AAA TAG TCC TG Nkx2.5 ACC TTT CTC CGA TCC GCG TTA GCG CAC TCA 28 58ATC CCA CTT CTT TAA TGG Mlc2a AAG GGA AGG GTC CCA AAC AGT TGC TCT ACC 3058 TCA ACT TCA TCA GCA GGA Mlc2v ACT TCA CCG TGT TCC TCC GTG GGT AAT GAT32 58 TCA CGA TGT GTG GAC CAA ANF TTCCTCGTCTTGGCCTTTT CCTCATCTTCTACCGGCA32 58 G TCTTC Nrg1 CCA ATG GCC ACA TTG AGC CTG GCC TGT AAT 32 58CCA ATA GGT TCT TCC TGT β-Actin GAA GGT GAC AGC ATT TTG GTC TCA AGT CAG27 58 GCT TCT GTG TGT AGA GGC

Continued stimulation with BMP-4 and BIO over days 2-5 further specifiedthe cells first as mesodermal, and later more specifically as cardiacforming mesoderm, as observed by expression of Isl-1 and Flk-1 (FIG.29). In the third window of treatment, BMP-4 and BIO were replaced byDkk-1, and Isl-1 expression increased (FIGS. 29, 30C) and Nkx2.5 (FIGS.29, 30D) and MEF-2c (FIGS. 29, 30E) are expressed. After a total of 5days of treatment with Dkk-1, the first markers of mature cardiomyocytesbegan to be expressed (FIG. 29).

At this point, we found further maturation was facilitated by replatingthe cells onto gelatin coated plates in basal medium, without usingadditional growth factors. Colonies of cells began to spontaneouslycontract after approximately 6 days and express cardiac specific genessuch as Troponin T, αMHC, and MLC-2v (FIGS. 29, 31, and 32).

We determined that contracting cardiomyocytes could subsequently bemaintained in serum-free medium for over one month.

A Small-Scale Screen in a Serum-Free, Monolayer Condition

Having identified the first serum-free monolayer condition for cardiacdifferentiation of ES cells, we sought to test its efficacy as aplatform to discover regulators of cardiomyogenesis. A collection ofapproximately 200 small molecules and growth factors reported to beinfluential in developmental processes were assembled. R1 ES cells weregrown and plated for differentiation on Matrigel in 24-well plates usingthe media and growth factors described above. The screening collectionwas then added and removed from each well at specific time points inorder to account for different developmental time windows. Thecollection was screened at days 0-2, 2-5, and 5-7.

Calcium Signaling Stimulates Mesodermal Differentiation

A strong positive stimulator of cardiac differentiation was found to bethe L-type Calcium channel (LTCC) agonist BayK 8644 (FIG. 33). Calciumsignaling is involved in several developmentally important signalingpathways. In regards to mesoderm formation and patterning, gradients ofintracellular Ca²⁺ and inositol-1,4,5-trisphosptate (Ins(1,4,5)P₃) havebeen suggested to play a role in formation of the dorsal-ventral (D-V)axis of the embryo.

The members of the Wnt-1 class signal through the canonicalWnt/beta-catenin signaling pathway, in which a stabilization ofcytoplasmic beta-catenin allows beta-catenin to regulate gene expressionin the nucleus. Conversely, members of the Wnt-5A class, stimulate therelease of intracellular Ca²⁺ and activate two Ca²⁺-sensitiveenzymes—Ca²⁺/calmodulin-dependent protein kinase II (CamKII) and proteinkinase C (PKC)—in a G-protein-dependent, but β-catenin-independent,manner. This pathway is commonly referred to as the Wnt/Ca²⁺ pathway, ornon-canonical Wnt signaling, as Calcium is thought to stimulate CamKIIand PKC.

To further ascertain the mechanisms by which calcium might bestimulating cardiac differentiation, growth factors and small moleculesin related pathways were tested. As stated, non-canonical Wnt signalingis dependent upon calcium. The model of differentiation proposed,however, states that canonical Wnt signaling is required at the earlystages differentiation. As canonical and non-canonical Wnt signaling aregenerally opposing signals, it is unlikely that calcium was operating inthe non-canonical pathway here. Nonetheless, Wnt5a was tested for itsability to stimulate cardiac differentiation at the same stage as BayK8644, both in conjunction with Bmp-4 and BIO and alone. Wnt5a treatmentresulted in a 20% decrease in overall beating efficiency in thedifferentiation, possibly by interfering with canonical Wnt signaling.

To clarify that the effect seen with BayK 8644 was indeed mediated bycalcium, several beta adrenergic regulating compounds were tested. Betaadrenergic receptors interact with G-proteins that contain the Galphas(Gs) and Galphai (Gi) subunits. The Galphas and Gbetagamma subunitspositively regulate L-type Ca²⁺ channels. Additionally, several isoformsof adenylate (adenylyl)cyclases are activated by Galphas subunits and assuch, activation of b-adrenergic receptors typically can elevate levelsof cyclic AMP (cAMP). Interestingly, the Gs activator isoproterenoldisplayed an even more potent cardiac inducing effect than BayK 8644,while the beta blocker propranolol greatly decreased cardiac potential.To test whether the calcium effect might be explained by cAMP levels, wetested an inhibitor of adenylyl cyclase 2′,5′-dd-3′-AMP-bis(t-Bu-SATE)and saw a decrease in the percentage of beating clusters. Additionally,the stable cAMP analog dibutyryl cAMP, often used to stimulate PKA/PKCsignaling, increased the percentage of beating colonies (FIG. 34).

To ascertain what role calcium and cAMP might be playing in this earlystage of cardiac differentiation, FACS was carried out using two markersinvolved in mesodermal fates. Specifically, a Brachyury-EGFP ES cell (agift from the Keller lab) was used to identify cells progressing to aprimitive streak type population. Further, cells were stained using aFlk-1 antibody to further delineate axial location of the primitivestreak type cells. Briefly, cells were treated with small molecules forday 0-2, and harvested at day 4.5 for analysis. As seen in the FACSplots (FIG. 35), the tested compounds had less of an effect onBrachyury-GFP expression, and more of an effect on Flk-1 expression.BayK8644, isoproterenol, and dibutyryl cAMP all displayed a similarincrease in the portion of GFP+ cells that were also positive for Flk-1,whereas propanolol decreased the percentage Flk-1 positive cells. Theseresults indicate that calcium and cAMP signaling is helping to furtherspecify the differentiation to a subset of the primitive streak that iscardiogenic (Flk-1+).

To further analyze the mechanism of action of the cardiogenic effectseen with these molecules, QRT-PCR was carried out on cells treated fromday 0-2 and harvested at day 4.5.

As seen in the expression analysis (FIG. 36), BayK8644, dibutyryl cAMPand isoproterenol all increased expression of Brachyury T, Flk-1, GATA-4and MESP1.

GATA-4 is a GATA family zinc-finger transcription factor that interactswith cardiac transcription factors Nkx2.5, Tbx5, and HAND2 and itsexpression is slightly earlier than that of Nkx2.5, though it is notcompletely specific for cardiac fates. The Mesp1 gene encodes the basicHLH protein MesP1 which is expressed in the mesodermal cell lineageduring early gastrulation. Disruption of the Mesp1 gene leads toaberrant heart morphogenesis, resulting in cardia bifida. MesP1 isexpressed in the heart tube precursor cells and is required formesodermal cells to depart from the primitive streak and to generate asingle heart tube. Flk-1 is a receptor for vascular endothelial growthfactor (VEGF) and marks both cardiac and hematopoietic precursors thatarise from the primitive streak.

A TGF-Beta Inhibitor Stimulates Cardiac Differentiation in Later Stagesof Development

The transforming growth factor (TGF)-beta superfamily is a diverse groupof cytokines that have pleiotropic effects in numerous biologicalprocesses. TGF-beta signaling is achieved via heteromeric complexes ofboth type I and type II serine/threonine kinase receptors. Upon bindingof the TGF-beta ligand and heterodimerization, the type I receptor isphosphorylated by the type II receptor which activates a signaltransduction cascade involving the Smad pathways. The superfamilymembers are classified into 2 categories: TGF-beta/Activin/Nodal, andBMP/GDF. The TGF-beta/Activin/Nodal group activates Activinreceptor-like kinase (ALK) 4, 5, and 7, which phosphorylate Smad2 and 3,while the BMP/GDF group activates ALK 1, 2, 3, and 6, which in turnphosphorylate Smad1, 5, and 8.

TGF-beta signaling is thought to be essential to the development ofmesoderm, in particular to the induction of the primitive streak. In invitro studies, the Keller group demonstrated dramatic reduction incardiac differentiation when EBs were treated with the TGF-betainhibitor SB431542. Additionally, as the primitive streak migratesinward in the developing embryo, the cells undergo EMT, enabling theirmotility; EMT is stimulated by TGF-beta signaling. Interestingly, wefound that treatment with the TGF-beta inhibitor A83-01 (FIG. 37)actually induced a higher number of beating colonies when used in days 5though 7 of the differentiation program. A83-01 is selective for theALK4, 5, and 7 receptors, and is more specific than SB431542.

To further demonstrate that inhibition of Activin/Nodal/TGF-betasignaling is playing a role in cardiac determination, the growth factorsActivin and TGF-beta were tested. As expected, treatment with thesegrowth factors resulted in a lower number of beating colonies, as isshown in FIG. 38.

QRT-PCR was carried out to observe the induction of cardiomyogenesis atthe transcript level. At day 5, when A83-01 treatment begins, thepredominant state is that of a developed primitive streak typepopulation that is yet to display robust expression of markers ofcardiac precursors such as Nkx2.5, Isl-1, and c-kit. As can be seen inFIG. 39, treatment with A83-01 from days 5-7 induced higher levels ofexpression of cardiac specific markers when assessed at day 8, whereastreatment with Activin A had the opposite effect.

Discussion

The model of cardiomyocyte differentiation we propose is therefore mostsimply modeled as involving 4 phases of differentiation, followed by aperiod of maturation. At the earliest stages, both BMP-4 and Wnt alonecan specify ES cells towards mesendodermal and mesodermal lineages. Whenboth pathways are activated concomitantly, the differentiation becomesmore specific to a subset of cardiac forming mesoderm. β-catenin inducesmesoderm formation through Brachyury T expression, and BMP-4/Smad1 isalso thought to induce mesoderm, thought its mechanism is unclear.

Continued BMP and Wnt signaling into the second phase of differentiationaids in specification of the cardiac fate through direct promotion ofcardiac precursor genes. The downstream pathway components of BMP-4,Smad1/4 activate Nkx2.5 expression through direct binding of itspromoter. Similarly, canonical Wnt signaling has been shown to inducedorsal mesoderm formation and promote formation of cardiac precursors bydirectly regulating expression of the cardiac precursor gene, islet-1.At the onset of the third stage of differentiation, the earliest markersof cardiac precursors are just beginning to be expressed. Specificationof cardiac precursors from this mixture of mesodermal cells was achievedwhen the Wnt inhibitor Dkk-1 was used.

After continued stimulation with Dkk-1 in the fourth stage ofdifferentiation the cells begin to express mature cardiomyocyte markersand form tight colonies that are resistant to trypsin mediateddissociation. We observed an increased efficiency in cardiac maturationwhen these colonies were detached and replated at a lower density ongelatin coated dishes, while being maintained in basal medium.

Though numerous growth factors have been implicated in cardiomyogenesis,we have found a minimal signaling requirement for cardiac specificationthat is remarkably simple. Our results indicate that BMP and Wntsignaling sit high in the order of mesodermal and subsequent cardiacspecification and that inhibition of Wnt signaling is important in laterstages of differentiation. It is inevitable that the regulation of morepathways is involved in specification of the cardiac lineages; thechemically defined, monolayer condition described herein should prove anexcellent tool in the pursuit of these factors.

While calcium/cAMP signaling is important in various differentiationprograms, its particular importance in the specification of cardiogenicmesoderm was previously unknown. This phase of differentiation coincideswith the inward movement of gastrulation which naturally involves anincrease in calcium concentrations. However, the only role calcium wasthought to play was in the motility and morphogenic changes that occurduring gastrulation. In our studies, it was demonstrated that calciumalso plays a signaling role in a cAMP pathway to direct cells toward acardiogenic mesodermal fate.

While monolayer conditions for the differentiation of neural andendoderm derivatives have been described, to our knowledge, similarsuccess has yet to be achieved in cardiac differentiation. Use of asimilar basal differentiation condition should facilitate comparisons ofdifferent ESC derived populations. Previously, BIO and BMP-4 have beenreported to stimulate self-renewal of mESCs. In this study, a novelcardiac mesoderm induction effect of BIO on ESCs was identified, and astrong positive role for BMP-4 in cardiac mesoderm formation wasobserved. Clearly, the effect of these molecules is contextuallydependent upon factors such as the composition of signaling molecules inthe medium and the extracellular matrix. As such, the present studyhighlights the value of the serum-free and monolayer approach to thestudy of ESC differentiation. Additional analyses of the regulation ofcardiomyogenesis should be facilitated by use of the described system asthe lack of undefined medium components should prove to produce morereliable and consistent results. Similarly, the use of a monolayerdifferentiation procedure allows for finer tuned mimics of the in vivodevelopment process.

Methods

Knock-in ESC generation: R1 ES cells were grown on feeder layer to nearconfluency, tryspinized, resuspended in 0.2-0.5 ml PBS, and transferredto an electroporation cuvette with 0.4 cm gap. The linearized targetingconstruct (25-60 ug) was added to the cuvette and briefly mixed bypipette and placed on ice for minutes before electroporation. A BioRadGene Pulser II was used at max capacitance and 500V. The cuvette wasimmediately iced for 10 minutes. The mixture was resuspended in warm ESmedia, and plated on feeder layer (1 6-well plate). The following day,media was changed and selection began with 0.1 ug/ml puromycin. Whencolonies became clearly visible to the naked eye, approximately one weeklater, colonies were picked. Colonies exhibiting round colony shape anddistinct borders were picked using a 200 ul pipette, containing 30 ultrypsin, and transferred into 96 well plates. Cells were then split into96-well plates for freezing, and 24-well plates for DNA harvesting. DNAwas harvested by adding sarcosyl lysis buffer and incubating overnight.The lysis was transferred into tubes containing 5 ul of proteinase K andmixed. Tubes were then filled EtOH and mixed until precipitate formedand centrifuged for 2 minutes at maximum speed. The supernatant wasremoved and another 500 uL of 70% EtOH was added and vortexed. Afteranother 5 minute centrifuge at max rpm, the supernatant was removed andthe tubes were allowed to air dry, before resuspending the pellet in 125ul water.

A primer set specific for the Nkx2.5 promoter and the GFP insert weredesigned to be used to test for correct integration of the vector. PCRreactions were run for all 500 clones generated, with 10 of the clonesshowing the correct PCR product size on an electrophoresis gel. The 10cell lines with the correct GFP insertion into the Nkx2.5 locus wereexpanded for testing. Hanging drop embryoid bodies were generated foreach cell line and monitored daily for GFP expression.

Embryonic stem cell maintenance: All reagents are from Invitrogen unlessotherwise stated. R1 mESCs are cultured on 0.1% gelatin-coated disheswith irradiated MEF (CF-1) cells in growth medium (ESGM). ESGM containsKnockout DMEM, 15% KO-Serum Replacement, 1×L-Glutamax, 1 mMbeta-mercaptoethanol, 1× non-essential amino acids, 1× nucleosides, and1000 U/ml LIF (Millipore). All reagents are from Invitrogen unlessotherwise stated. Prior to differentiation, cells were passaged once onlow-density MEF.

Cardiac differentiation: Cells are plated at a density of 200,000cells/well of a 6-well plate in 2.5 ml of ESGM. 6-well plates are coatedovernight at 4° C. with 1 ml of Matrigel (BD Bioscience) diluted 50×from stock concentration. 24 hours after plating, or after sufficientcolony size was achieved, cells are switched into chemically definedmedium (CDM) and treated with 4 uM BIO (Calbiochem) and 20 ng/ml BMP-4(R&D). CDM contains RPMI 1640, 0.5×N2, 1× B27 (without Vitamin A), 0.5×Glutamax, 0.55 mM beta-mercaptoethanol, 1× non-essential amino acids, 1×nucleosides. At Day 2, medium is changed, and again CDM containing 4 uMBIO with 20 ng/ml BMP-4 is added to the cells. At Day 5, cells arewashed once with PBS and medium is changed to CDM containing 100 ng/mlDkk-1 (R&D). At Day 7, medium is changed, and again CDM containing 100ng/ml Dkk-1 is added to the cells. At Day 10, cells are trypsinized(0.05% trypsin) (Approximately 5 minutes at 37° C., or until cells beginto detach) and replated onto gelatin coated 6-well plates in CDM with noadditional growth factors. By this time, the cells will have formedtight colonies that will be difficult to disrupt (Supplementary FIG. 2b). If the cells do not detach with trypsin alone, a cell scraper can beused. The trypsin is used primarily to detach the cells rather than todissociate them to a single cell suspension. It is preferable for thecolonies to remain intact. The trypsin is briefly quenched with MEFmedia, cells are centrifuged, and resuspended in CDM for plating. Mediumis refreshed as needed to remove dead cells and debris (every 1 to 2days).

Immunofluorescence: Cells were fixed in 4% paraformaldehyde (Sigma) for15 minutes, and stained in 0.3% Triton (Sigma) containing 5% donkeyserum (Jackson ImmunoResearch). Antibodies were used at dilutions ofMF20 [Developmental Studies Hybridoma Bank (DSHB)] 1:100, CT3 (DSHB)1:100, Nkx2.5 (Santa Cruz) 1:200, MEF-2c (Santa Cruz), Islet-1 (DSHB)1:100, and Brachyury T (Santa Cruz) 1:200. Secondary antibodies (JacksonImmunoResearch) were used at 1:200 for Cy2 and 1:500 for Cy3. Nuclei arestained with DAPI (Sigma).

FACS analysis: Cells were harvested into a single cell suspension bytreatment with 0.05% trypsin, quenched with serum containing medium, andresuspended in FACS buffer: 1×PBS (Ca/Mg²⁺ free), 1 mM EDTA, 25 mM HEPESpH 7.0, 1% Fetal Bovine Serum. Cells were then filtered with 0.2 μmfilter, and stored at 4° C. until analysis.

qRT-PCR: Total RNA from each sample was prepared from 5×10⁴ cells usingRNeasy mini kit (Qiagen). Reverse transcription was carried out by usingReverse Transcription System (Promega) and PCR performed using PCR SuperMix (Invitrogen).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of converting an animal cell from a first non-pluripotent cell fate to a second non-pluripotent cell fate, the method comprising: increasing the quantity of at least one reprogramming transcription factor in an animal cell having the first cell fate to generate a cell that differentiates in response to lineage-specific differentiating factors to a second cell fate different from the first cell fate; and submitting the cell that differentiates in response to lineage-specific differentiating factors to conditions that induce differentiation of the cell to generate a cell having the second non-pluripotent cell fate.
 2. The method of claim 1, wherein the increasing step comprises limiting the development or growth of pluripotent cells.
 3. The method of claim 2, wherein the development or growth of pluripotent cells is limited by limiting the expression of the reprogramming transcription factor.
 4. The method of claim 2, wherein the development or growth of pluripotent cells is limited by contacting the cell that differentiates in response to lineage-specific differentiating factors with an inhibitor that inhibits the growth of pluripotent cells.
 5. The method of claim 4, wherein the inhibitor that inhibits the growth of pluripotent cells is a JAK inhibitor.
 6. The method of claim 1, wherein the cell that differentiates in response to lineage-specific differentiating factors does not significantly express Nanog.
 7. The method of claim 1, wherein the method occurs in the absence of exogenous LIF.
 8. The method of claim 1, wherein the cell that differentiates in response to lineage-specific differentiating factors is not capable of forming a teratoma.
 9. The method of claim 1, wherein the method does not comprise isolation or selection of cells between the increasing and submitting steps.
 10. The method of claim 1, wherein the cell that differentiates in response to lineage-specific differentiating factors is not an induced pluripotent stem cell.
 11. The method of claim 1, wherein the animal cell having the first cell fate is a fibroblast or a neural precursor cell.
 12. The method of claim 1, wherein the cell having the second non-pluripotent cell fate is a cardiomyocyte.
 13. The method of claim 12, wherein the submitting step comprises contacting the cell that differentiates in response to lineage-specific differentiating factors with BMP4, a calcium channel agonist, a Gαs activating agent, a cAMP analog, and/or a GSK-3 inhibitor.
 14. The method of claim 1, wherein the cell having the second non-pluripotent cell fate has a neuronal cell fate.
 15. The method of claim 1, wherein the cell having the second non-pluripotent cell fate has a pancreatic cell fate.
 16. The method of claim 1, wherein the reprogramming factors are selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc polypeptide.
 17. The method of claim 1, wherein the reprogramming factors comprise an Oct polypeptide, a Klf polypeptide, and a Sox2 polypeptide.
 18. The method of claim 1, wherein the reprogramming factors comprise an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc polypeptide.
 19. The method of claim 1, wherein the time from initiation of the increasing step to the generation of the cell having the second non-pluripotent cell fate is no more than 25 days.
 20. The method of claim 1, wherein the time from initiation of the increasing step to the generation of the cell having the second non-pluripotent cell fate is no more than 20 days.
 21. The method of claim 1, wherein the time from initiation of the increasing step to the generation of the cell having the second non-pluripotent cell fate is no more than 15 days.
 22. The method of claim 1, wherein the time from initiation of the increasing step to the generation of the cell having the second non-pluripotent cell fate is between 10-22 days.
 23. The method of claim 1, wherein the conditions that induce differentiation are chemically defined conditions.
 24. The method of claim 1, wherein the increasing step is performed in vivo.
 25. The method of claim 1, wherein the method is conducted at least partly in vivo.
 26. The method of claim 25, wherein the submitting step occurs by inducing the increasing step in a cell in a cellular context in vivo such that endogenous signals cause the cell to have the second non-pluripotent cell fate.
 27. The method of claim 1, wherein the method is conducted in vitro.
 28. A method of differentiating an animal cell into a cardiomyocyte, the method comprising: contacting the animal cell with a GSK-3 inhibitor and/or a BMP protein under conditions to generate a cardiomyocyte.
 29. The method of claim 28, wherein the animal cell is a non-human animal cell.
 30. The method of claim 28, wherein the animal cell is a human cell.
 31. The method of claim 28, wherein the cell is a pluripotent cell.
 32. The method of claim 28, wherein the method is conducted at least partly in vivo.
 33. The method of claim 28, wherein the method is conducted in vitro.
 34. The method of claim 1, wherein the conditions are chemically defined conditions.
 35. A method of transdifferentiating an animal cell into a cardiomyocyte, comprising: introducing into a non-pluripotent animal cell having a first non-pluripotent cell fate one or more polynucleotides encoding one or more polypeptides selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc polypeptide; or contacting a non-pluripotent animal cell having a first non-pluripotent cell fate with one or polypeptides selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc polypeptide; and contacting the non-pluripotent animal cell with a GSK-3 inhibitor, a calcium channel agonist, a Gαs activating agent, a cAMP analog, and/or a BMP protein under conditions to generate the cardiomyocyte; thereby differentiating the non-pluripotent animal cell into the cardiomyocyte.
 36. The method of claim 35, wherein the method further comprises contacting the non-pluripotent animal cell with a JAK inhibitor.
 37. The method of claim 36, wherein the non-pluripotent animal cell is contacted with the JAK inhibitor prior to contacting the cell with the GSK-3 inhibitor, the calcium channel agonist, the Gαs activating agent, the cAMP analog, and/or the BMP protein.
 38. The method of claim 36, wherein contacting with the JAK inhibitor comprises culturing the non-pluripotent animal cell in the presence of the JAK inhibitor.
 39. The method of claim 38, wherein the non-pluripotent animal cell is cultured in the presence of the JAK inhibitor for a period of about one to about nine days.
 40. A method of transdifferentiating an animal cell into a neural progenitor cell, the method comprising: introducing into a non-pluripotent animal cell having a first non-pluripotent cell fate one or more polynucleotides encoding one or more polypeptides selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc polypeptide; or contacting a non-pluripotent animal cell having a first non-pluripotent cell fate with one or polypeptides selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc polypeptide; and contacting the non-pluripotent animal cell with FGF2, FGF4, and EGF under conditions to generate the neural progenitor cell; thereby differentiating the non-pluripotent animal cell into the neural progenitor cell.
 41. The method of claim 40, wherein the method further comprises contacting the non-pluripotent animal cell with a Wnt inhibitor, a JAK inhibitor, or both a Wnt inhibitor and a JAK inhibitor.
 42. The method of claim 41, wherein the non-pluripotent animal cell is contacted with the Wnt inhibitor, the JAK inhibitor, or both the Wnt inhibitor and the JAK inhibitor prior to contacting the cell with FGF1, FGF4, and EGF.
 43. A method of transdifferentiating an animal cell into a retinal pigmented epithelium cell, the method comprising: introducing into a non-pluripotent animal cell having a first non-pluripotent cell fate one or more polynucleotides encoding one or more polypeptides selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc polypeptide; or contacting a non-pluripotent animal cell having a first non-pluripotent cell fate with one or polypeptides selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc polypeptide; contacting the non-pluripotent animal cell with FGF2, FGF4, and EGF under conditions to generate a neural progenitor cell; and contacting the neural progenitor cell with a TGFβ inhibitor and a GSK-3 inhibitor to generate the retinal pigmented epithelium cell; thereby differentiating the non-pluripotent animal cell into the retinal pigmented epithelium cell.
 44. A method of transdifferentiating an animal cell into a tyrosine hydroxylase (TH)-positive neuronal cell, the method comprising: introducing into a non-pluripotent animal cell having a first non-pluripotent cell fate one or more polynucleotides encoding one or more polypeptides selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc polypeptide; or contacting a non-pluripotent animal cell having a first non-pluripotent cell fate with one or polypeptides selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc polypeptide; and contacting the non-pluripotent animal cell with FGF8 and SHH under conditions to generate the TH-positive neuronal cell; thereby differentiating the non-pluripotent animal cell into the TH-positive neuronal cell.
 45. A method of transdifferentiating an animal cell into a pancreatic lineage cell, the method comprising: (1) introducing into a non-pluripotent animal cell having a first non-pluripotent cell fate one or more polynucleotides encoding one or more polypeptides selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc polypeptide; or contacting a non-pluripotent animal cell having a first non-pluripotent cell fate with one or polypeptides selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc polypeptide; and (2) under conditions to generate the pancreatic lineage cell, (a) contacting the non-pluripotent animal cell with a TGFβ/Activin/Nodal family member and a JAK inhibitor; (b) contacting the cell of step (2)(a) with a TGFβ/ALK5 receptor inhibitor, BMP4, bFGF, and RA; and (c) contacting the cell of step (2)(b) with nicotinamide; thereby differentiating the non-pluripotent animal cell into the pancreatic lineage cell.
 46. A method of transdifferentiating an animal cell into an induced definitive endoderm (iDE) cell, the method comprising: introducing into a non-pluripotent animal cell having a first non-pluripotent cell fate one or more polynucleotides encoding one or more polypeptides selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc polypeptide; or contacting a non-pluripotent animal cell having a first non-pluripotent cell fate with one or polypeptides selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc polypeptide; and contacting the non-pluripotent animal cell with a GSK-3 inhibitor, an HDAC inhibitor, and a TGFβ/Activin/Nodal family member under conditions to generate the iDE cell; thereby differentiating the non-pluripotent animal cell into the iDE cell.
 47. A method of transdifferentiating an animal cell into a pancreatic beta cell, the method comprising: (1) introducing into a non-pluripotent animal cell having a first non-pluripotent cell fate one or more polynucleotides encoding one or more polypeptides selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc polypeptide; or contacting a non-pluripotent animal cell having a first non-pluripotent cell fate with one or polypeptides selected from the group consisting of an Oct polypeptide, a Klf polypeptide, a Sox2 polypeptide and a Myc polypeptide; (2) contacting the non-pluripotent animal cell with a GSK-3 inhibitor, an HDAC inhibitor, and a TGFβ/Activin/Nodal family member under conditions to generate an iDE cell; and (3) under conditions to generate the pancreatic beta cell, (a) contacting the iDE cell with FGF7, RA, a Hedgehog pathway inhibitor, a BMP inhibitor, and a TGFβ/ALK5 receptor inhibitor; (b) contacting the cell of step (3)(a) with EGF and a Notch inhibitor; and (c) contacting the cell of step (3)(b) with bFGF and nicotinamide; thereby differentiating the non-pluripotent animal cell into the pancreatic beta cell.
 48. The method of claim 47, wherein step (3) further comprises contacting with extendin
 4. 